JPH01500591A - Virtually pure cytotoxic trigger factor - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Virtually pure cytotoxic trigger factor description technical field The present invention provides T! II follicle induction and especially mono primed, peripheral blood mononuclear cells, and act on tumor cells. The present invention relates to human proteinaceous lymphocytes that induce cytotoxicity.

background Cells of the monocyte/macrophage lineage have antibacterial effects, mediate cytotoxicity, aggregation and initiation of the fibrinolytic and fibrinolytic protease pathways, complement protein synthesis and IL-1 (IL-1) and tumor necrosis factor alpha (TNF-A). Possesses a variety of effector abilities, including the secretion of immunologically important monokines. There is. These states of cellular activation and molecular products can be induced by appropriate signals. It will be done. Cohn (1978), Journal of Immunology (J, Immunol,) Volume 121, page 813, North (1 981) LyIIIphokines, vol. 3, p. 1, a. Adams et al. (1984) Annual Review of Immuno. logy (Ann, Rev, f+a+uno1.) 2 @, 283 pages, Nishinto Edgington et al. (1985) Mononuclea hupgosite (M ononuclear Phagocytes) % pp. 687-696, Vasalli et al. (1977) Journal of Experimenta Le Medicine (J, Exp, Med,) Vol. 145, p. 429, Liftman (l (1977) Journal of Experimental Medicine Dishin (J, Exp, Med,) vol. 145, p. 1344, and Dinarello ( Dinarello) (1984), Revue of Infectious ・See Rev, Infect, Dis, volume 6, page 51.

These cellular functions are of the contact- or lymph-in-mediated type, selected Coupled with the immune response through cooperative interactions mediated by T lymphocytes . These responses are not themselves antigen-specific, but [Nathan (1983) Journal of Experimental Medicine (J,E xp, Med, ) vol. 158, p. 670, Gregory et al. (19 1985) Journal of Clinical Investigation (J, Cl1 n.

Invest, ) vol. 76, p. 2440, Herrmann et al. 1985), Journal of Experimental Medicine (J,E xp, Med, ) volume 162, page 1111, and Amento et al. (1985) Journal of Immunology (J, I+gsuno1.) 34'e, p. 350), but it is still central to the immunological response in the living body. T that in turn directs the expression of monocytic/macrophage effector functions. Immunologically induced into cells.

The study of human and murine monocytes and macrophages is the basis of the mononuclear phagocytic system. cells are physiologically in a dormant or latent state and are susceptible to tumor cell and pathogen destruction. consistently demonstrate that they do not normally develop an effective ability to Instead, A series of steps in which macrophages mature from blood monocytes to tissue migration is high in vitro (inν1tro) or in vivo (in vivo). required to express a highly developed response. as well as some unrelated agonists , one or more lymphocytes in a set are associated with monocyte/macrophage activation are doing. This substance is a component of the cellular immune effector response. Fiddler (1999). 1984), Lymphokine Re5ea rch) Volume 3, page 51.

Interferon-gamma (IFN-Q) affects monocytes and macrophages. Nathan et al. (1 983) Journal of Experimental Medicine (J, Exp, Med, ) Volume 158, page 670, Nathan et al. Journal of ・Experimental Medicine (J, Exp, Med,) Volume 160, 60 (page 0), IFN-G initiates a complex series of changes in macrophage function. The first molecularly determined T-phosphorus protein identified as MAF associated with the mining phase. It was proven to be a cell product. Also, IFN-G is MAF-1 here. It is called. Pace et al. (1983) Journal of Immunology J, lm1unol, vol. 130, p. 2011, Pace, etc. (1983) Proceedings in National Academy of Sciences Jens (Proc, Natl, Acad.

Sci, ) USA vol. 80, p. 3782, Fischer + - (Fischer), etc. (1983) Journal of Immunology (J, Immunol,) 1 Vol. 30, p. 1220, Le et al. (1983) Journal of Immuno. J, Immunol, Volume 131, Page 2821, Sakli k) et al. (1985) Cancer Research 45 je, p. 1940 and Svederski et al. (19 84U Journal of Experimental Medicine (J, Exp, Me d,) Volume 159, page 812.

A series of signals were detected in a variety of assays to identify activated phenotypic monomers attributable to MAF. It became clear at some point that it was necessary to induce cytotoxicity and macrophages. It was. MAF-l, which has been known from the beginning, acts as a priming factor, and A two-belief hypothesis has been proposed in which a second signal is responsible for the final trigger of activation.

Hibbs et al. (1977) Science 197 Volume 279, Ruco et al. (1978) Journal of Immunology - (J, lff1+*unol) Volume 121, Page 2035, Meltzer (Met zer) (1981), Lyenpho Kines 3 Vol. 319, and Schreiber et al. (1981) See Fed, Proc, Vol. 40, p. 1002.

In vitro (i To induce tumor-killing cytotoxicity in vitro, the endotoxin A second or trigger signal was indicated. Pace et al. (1983), Null of Immunology (J, Immunol,) 130'l, 2011 Pace et al. 1983) Proceedings in National ・Academy of Science (Proc Natl, Acad, Sci,) U S A, 80 S.

p. 3782, Hibbs et al. (1977) Science e) 197 e 279 pages, Ruco et al. (1978), Jhana Le of Immunology (J, ra++auno1.) Volume 120, Page 2035 , Mertzer (1981) Lymph Schreiber et al. (1 981) Fed, Proc, Volume 40, Page 1002, Urchin Weinberg et al. (1978) Journal of Immunology Logical Methods (J, Immunol, Methods) Volume 37, 225 Page, Schultz (1982) Journal of Inter Feron Research (J, Interferon Res,) Volume 2, page 459 , and Page et al. (1985) Journal of Leukemia See Biology (J. Lenk, Biol.), vol. 37, p. 475.

Listeria monocytogens killed by heat contact with the genus Ogenes was also sufficient.

Schreiber et al. (1982) Journal of E. Kusuberi Mental Medicine (J, Exp, Med,) 156f'677 pages , in addition, the high administration of lymphoid-containing medium requires the presence of a certain period of time, Trigger tumor cytotoxicity caused by IFN primed monocytes (macrophages) You can do it.

Hibbs et al. (1977) Science 19 Vol. 7, p. 279 and Meltzer et al. (1982), Fet. See Fed, Proc, Vol. 41, p. 2198.

Monocytes and macrophages as effector cells controlled by T cells The role of lineage cells remains unclear due to the presence of various agonists that induce direct responses It can become clear. Endotoxin such as LPS in vitro Only after being excluded from the analytical system (Weinberg et al. (1999) 1978) Journal of Immunology (J, Immunol,) Volume 121 p. 721), with at least two induction steps: priming and triggering. (Meltzer (1981)) Lymphokines, Vol. 3, p. 319). In Vipo (inv) ivo), Bacillus carmetschibnilin (Bacillus Ca CBCG) In addition, in vitro, T helper cells Induced lymphadenosis and subsequent stimulation by antigens or mitogens Priming is now complete.

Recently, IFN-G has been used to develop monocytogenes in vitro. was identified as a necessary and sufficient reagent for priming (macrophages). It was. Page et al. (1983) Proceedings in the Nation. National Academy of Science (Proc, Natl, Acad, 5ci ) USS A Volume 80, Page 3782. It's a priming lymph talent. The priming lymphoid preparation can be substituted with the IFNG preparation. If , the priming ability is lost. However, based on current data, if The priming activity of N-G is the l! of macrophages. Tumor killing ability or cytotoxicity There is a general opinion that it is insufficient to fully induce Tarameri (Tar (1980), Journal of Immunological Methods (J. Iaeunolo Methods) Volume 37, Page 225. available The data indicate that monocyte (macrophage) cells, as exemplified by endotoxin, The need for another or “secondary” signal to trigger cytotoxicity We are doing so.

Because endotoxins are significant contaminants of all culture media and serum, researchers Only with priming lymph talent like IFN-G only, Macropher Inadvertently concluded that dicytotoxicity could be directly activated (sea urchin inbarb). (Weinberg) et al. (1987) Journal of Immunology (J. I+amuno1. ), vol. 121, p. 72), “macrophage-activating factor” I ended up assigning IFN-G. This problem is caused by small amounts of endotoxins (e.g. LP). priming lymph in the presence of S) and a small amount of plastic. Monocytes (macrophages) against endotoxins in the presence of iming lymphocytes. It is more severe in cases of alternating exaltation of responses (e.g.). Pace (1981) Journal of Immunology (J, Immunol, ) 1261', 1863 pages.

Lymphoid or IFN-G alone inhibits monocyte (macrophage) cells. In contrast to the inability to activate cytotoxicity, relatively high concentrations of C1pg /■ and above) endotoxins can induce sufficient cytotoxicity without pre-briming. can be guided. Dos et al. (1978), Journal of Experimental Medicine (J, Exp, Med,) Volume 148, Page 544 and Ca+weron et al. (1980) Journal of Immunology. (J, r+u+uno1.) Vol. 124, p. 708). However, the monosite and the endotoxin concentration required for direct induction of cytotoxic function in macrophages. causing shock and disseminated vasotropic coagulation in vivo; Lllevitch et al. (1984) Fed, Proc, ) Volume 43, Page 2755.

In a published report (Kornbluth et al. (1985) ) Fed, Proc, Vol. 44, p. 1699), we Nuclear activity was demonstrated in IFNG-primed human peripheral mononuclear cells (PBM). triggers monocyte-mediated cytotoxicity in cultured human tumor cells. I reported that I found out. Its lymphatic activity is a molecule or group of molecules. It was not known whether this was due to a known or unknown coordinated action between the two. Discussion later The results discussed demonstrate that a single lymphin molecule, named CTF, Cytotoxic to primed (stimulated) monocytes such as clophages It is explained that a second trigger signal can be provided to induce the trigger.

U'-゛na, The present invention provides a substantially pure human protein designated cytotoxic trigger factor (CTF). Mixtures containing proteinaceous lymphoids, CTF, their preparation as well as their uses It was devised regarding the law.

Substantially pure CTF has an apparent relative content of about 6 to about 150 kilodaltons. Gel exclusion chromatography using a gel matrix that can fractionate small-sized proteins A protein with an apparent molecular weight of approximately 55 kilodaltons as determined by And, interferon gamma primed peripheral blood is from nuclear cells. , can secrete tumor cell killing factor III, including tumor netarosis factor alpha. can. Lymphoids are normally present (endogenously) when CTF is secreted. interferon gamma, interleukin-2 and direct cytotoxicity Like Syn, it is virtually free of pyrogens such as endotoxin. CTF is Differentiated homologous stimuli such as those exhibiting the T3"T4" T8-Ml-phenotype It can be secreted from normal human helper T cells.

Two additional substantially pure proteins are also contemplated. these proteins have substantially the same* potency and purity characteristics as the proteins described above, but When chromatographed on the same matrix as It exhibited an apparent relative molecular weight of 0 kilodaltons or more.

Another feature of the invention is that primed effectors such as human monocytes - induces cells to contain Il! containing tumor netarosis factor alpha! tumor cell killing factor This is a method of secreting it. Here, primed human effectors in aqueous medium - induces cells to secrete l1ljX cell killing factors, including tumor necrosis factor alpha. an aqueous composition comprising substantially pure CTF present in an effective amount to trigger mix with things. The tumor netarosis factor alpha obtained in this way may also be used. can be recovered.

Another feature of the present invention is that primed human thought about how to induce vector cells to mediate the death of tumor cells. It is something. Here, primed human cells such as PBM providing a monocytic source - Effector cells were isolated from primed effector to 1ilX in aqueous medium. Effective in triggering the secretion of tumor killing factors that induce netarosis factor alpha an aqueous composition containing an amount of substantially pure CTF. should be exterminated Tumor cells are contacted with secreted tumor killing factors.

In one embodiment of the above features of the invention, exemplary plating of the effector cell population Utilizing monocytes as primable and primable cells, the method ・Performed in vivo, and Primed Monosite is Tumor cells that are to be killed and will be present within the treated patient's tumor .

In one variation of the tree N, 341 of this feature of the invention, the priming in vivo, whereas in other variables Imaging is performed in vitro. In vivo (i n vivo) Priming is the activation of energy such as monocytes circulating within the patient's body. Administer IFN-G parenterally in an amount sufficient to prime effector cells. This can be done by in vitro primin For human leukocytes, a preparation of approximately 1X10' cells per milliliter of human leukocytes is used. entered in the same way as by Leukapharesis. hand and maintain the contact time for about 36 to about 72 hours. Approximately 200U/mj from 4! mixed and contacted with a priming amount of IFN-G. Priming by

The in vitro primed cells are then injected, etc. is introduced into the patient's body. The patient in vivo Or, in vitro primed monocytes are concentrated within the Lt tumor. (move and localize) and (those primed monosites that are already there) (in addition to) keeping the patient's blood free for a sufficient period of time. Water-soluble physiology in sufficient quantities to trigger concentrated primed monocytes A composition comprising substantially pure CTF in a biologically acceptable carrier. The concentrated drug is administered to the patient to secrete a TNF-A-containing tumor killing factor. Trigger imdo monosite.

In yet another embodiment, the method is performed entirely in vitro. ro) and the tumor cells contacted with the tumor killing factor are primed monocytogenes. Supplied externally within IO time after triggering.

The present invention provides several advantages and advantages.

One advantage is that it is a novel drug that can be used to induce tumor cell death. The goal is to provide a CTF with a lot of power.

One advantage of the present invention is that it selectively treats tumors without damaging other cells. The objective is to provide a method for killing cells.

Another advantage of the present invention is that it further provides tumor necrosis factor alpha. To provide a method for inducing primed monocytes to secrete .

Further, other advantages and advantages will be apparent to those skilled in the art from the detailed disclosure described later. Probably.

Brief description of the drawing FIG. 1 shows a series of graphs (panels A-E).

Panel A is a chronological diagram of the three-step verification procedure used in the present invention. Indicates the presence of cytotoxic trigger factor (CTF). Area on the left (open) - Round bottom of 966 microplates as a source of primable effector cells Peripheral blood mononuclear cells (e.g., T cells, B cells, natural killer cells, mononuclear cells, site, etc.), from 2 x 10' pieces/ml to 6.25 x 10' pieces/ml torr (or 5 x 104 cells/ml for purified blood monocytes) and IF 4 of this assay, maintained for 48 hours in the presence of NG150 units/ml. Showing an 8 hour in vitro briming step .

The first vertical line on the right of center indicates the cytotoxic activity of prime-capable cells (stage ■). To trigger, add Lymphokine-containing medium or other cytokines to each well. The time for mixing 50-100 μl of the sample preparation is shown. After that mixing, the pre Return the substrate to the incubator. This triggering stage lasts for 6 hours, This is represented by the stippled area in the figure.

The vertical line to the right of the above vertical line represents the target (“Cr-labeled) actinomycin (Act ) 50μl of D-treated WEH1164 cells (2X10ゝ/mJ) was treated with IFN-G. Third lysis stage (stage ■) mixing with primed and triggered cells It shows the start of.

The space between the last mentioned vertical line and the rightmost vertical line (shaded area) is the target area. In vitro for 6 hours during which monocyte-dependent lysis of the cells occurs. ) represents the dissolution stage.

The right-most vertical line represents the time to stop the assay, allowing for uptake due to cell lysis. The amount of 5IC released into the supernatant is measured and indicates the presence of CTF.

For cell lysis, remove one-half of the supernatant and add it to scintillation cocktail. and count per minute in a liquid scintillation counter for 1 minute. Measure numbers. Data are described in Materials and Methods, Section V(E). It is reported as “% specific release capacity”.

The graphs in Panel B%C%D and E were calculated using the test procedure described for Panel A. Cytotoxicity of mushrooms or their ablation or alterations as mentioned. Ru. Each vertical axis indicates cytotoxicity units (% specific release capacity). Each horizontal axis represents the existing Effects on target cells (“Cr-labeled Acrylic D-treated WEH1164 cells)” The ratio E:T of vector cells (PBM for the analyzes shown) is expressed in units of ing.

Panel B shows the effect of LPS triggering. First set of points (○-O ), PBMs incubated with medium alone for 48 hours were found to contain spontaneous monocytes. mediated cytotoxicity. The second set of points (Δ-Δ) is 48 time, PBM incubated in medium also containing 150 U/■l of IFN-γ , did not exhibit spontaneous monocyte-mediated cytotoxicity. 3rd cent point (・-・ ) was supplemented with 10 micrograms/milliliter LPS for 48 hours, IF PBM incubated in medium containing 150 U/val of N-γ Significant cytotoxicity was mediated during the 6 hour lysis stage of the assay. This test method is , allowing the identification of primed monocytes/macrophages, monocytes/macrophages that are not present can be primed by IFN-γ. Which indicates that.

Panel C is induced by lymphin-containing supernatant (LK-3N) under three conditions. Trigger ring shown. The first cent point (O-○) is the 48-hour incubation point. Incubator underground (described in Materials and Methods, Section V(A)) (unprimed), then incubated in LKSN for 6 hours. BM is shown to be unable to induce cytotoxicity.

The second cent point (・-・) is 150 U/tal (D I F N -6 hour ink of PBM primed by pre-incubation with G. tion has been shown to induce significant monocyte cytotoxicity. Third The set point (Δ-Δ) is 48 for LK-3N, which includes both IFN-G and CTF. Time B M - [-7 When incubated with Efter, monocytic cells It has been shown to induce toxicity.

Panel D shows control supernatant (Co-SN, non-activated human PBM) under three conditions. (derived medium). this panel The graph shows that triggering and lymph incubation was performed using cultured non-primed PBM or is not a constituent product of cultured Daudi cells used to activate PBM. It is shown that. The first cent point (O-○) indicates effector cells for 48 hours. Incubate in medium then 6 hours c.

-Demonstrates lack of cytotoxicity when incubated in 3N. 2nd cent Points (・-・) indicate effector cells were treated with 150U/+wj! for 48 hours. 0IFN Incubate and test in G.

= indicates lack of cytotoxicity when incubated with SN for 6 hours. 3rd period Points (Δ-Δ) indicate effector cells incubated with Co-5N for 48 hours. shows the lack of cytotoxicity when incubated.

Panel E shows that activation of lymphokine produces triggering lymphokines. It explains things. Is the first cent point (0-O) a commonly used PBM? 10 nanograms/milliliter of phorbol from nylon wool enriched T cells ・Myristate acetate (PMA) and 1 micromolar (μM) calci Efficacy of chemically activated ionophore A23187 with a 3-hour pulse. It shows results. After 48 hours of activation incubation, the supernatant medium was diluted 10x. Concentrate to I took it. Although it is less than briming with IFN-G, this amount (PMA /Cal-3N) in the medium for 48 hours in the absence of IFN-G briming. Induced monocytic cell activity in PBM maintained in PBM. (This result is based on PMA Note that the second set is affected by the presence of carryover materials and contaminating LPS. The points (・-・) indicate 48-hour incubation with 150 U/a+1 IFN-G in the medium. cuvation and subsequent PMA/CaI as a second signal Effector briming is possible after 6 hours of incubation with SN. This has been shown to significantly increase cytotoxicity. The third cent point (△-△) is P The cytotoxicity shown after 6 h incubation with MA/Ca1-3N was 48 hour incubation with MA/Car-3N showed a decrease in There is.

Figure 2 shows the cytotoxicity when using PBM as the effector cell source. Demonstrates the function of ll1X necrosis factor alpha (TNF-α, TNF-A) There is. Panel A shows that the increase in TNF-α when mixed with IFN-G-containing medium was Cytotoxic to ActD-treated WEH1164 target cells in a 6-hour lysis assay. It shows that it induces. The EAT ratio is 50:1.

The horizontal axis shows the cytotoxicity units (% specific release capacity), while the vertical axis shows the milliliter equivalent. The figure shows the added TNF-α unit.

Panel B shows the monoclonal monoclonal that binds and neutralizes TNF-A. When antibodies are present in the mixture, IFN-G primed mixed with LK-3N PBM-induced cytotoxicity (% specific release capacity) is shown to be reduced. E.A. The T ratio is 50:1.

The vertical axis shows inhibition by increasing amounts of monoclonal antibody added per milliliter. TNF-A activity unit (INF inhibited by MAb/1ml) −α unit).

Figure 3 shows the plying of PBM as effector cell-containing cells by IFN-G. Two panels showing the kinetics (temporal study) and dose response (dose study) of Contains files. Here, the dosage and time requirements are 50:1 PBM:i A constant 30% cell ratio was used for 6 hours of stage induced by the component LK-3N (LK-SN added in the last 6 hours). The cytotoxic effects of IFN-G on priming were evaluated.

As seen in panel A, a fixed amount (150 U/yal) of recombinant IFN -G (r　I　F　N　-1) I was at my best when I was in college.

The results in panel B are shown when the amount of recombinant I FN-G was varied (at 48 hours). Using 48 hour briming of rI FN-r, U/mj added to This is what was obtained. Minimal priming is obtained at approximately 4-20 U/m6. , while maximum priming is obtained above 20 U/m1, and beyond this The 0 dotted line (-...-), which was relatively constant in the upper range, becomes thin with IFN alone. This shows that it does not induce cell activity.

Figure 4 consists of one graph and shows the triggering of cytotoxicity by CTF. The time requirements for this are shown below.

PBM primed by incubation with IFN-G for 48 hours. The CTF-containing composition ( ・-・i　LK-5N, and 〇-〇PMA/Cat-3N) were contacted and maintained. . Regardless of its origin, the data indicates that triggering is rapid. It shows.

FIG. 5 is a graph showing a method for quantifying CTF. A series of CTF-containing preparations Add the dilution of and the culture so prepared was exposed to 150 U/m6 of I for 48 hours. PBM primed with FN-G was used with an E:T ratio of 50:l for 6 hours. Maintained. CTF was quantified as half-maximum units.

CTF quantitative values varied significantly depending on the method used for production. LK- The 3N preparation contains 100 U/lllff1 or more (1) IF) It-Q, On the other hand, the PMA/A23187 fraction lacks IFN-G according to the RIA assay. was.

Figure 6 shows one of the induction requirements for the production of CTFs by T cell clones. This is a graph of Specifically responds to HLA-DR alloantigen of irradiated Daudi cells. human T helper clones from normal donors at a T:Dauzi ratio of 1:4. Co-cultured with irradiated Daudi cells. The culture medium supernatant was collected intermittently to determine the rate of CTF production. The degree theory was analyzed (-Dowsey activation clone 62;・Dowsey activation clone 2 5) Control supernatants are independent of irradiated Dauzi cell activation and T cell clones. Cultures were performed in parallel, and T cells and Daudi cell supernatant were mixed at a ratio of 1:4 before use. (Ro control clone 62; ○ control clone 25).

The results shown in the graph show that co-culture of irradiated Daudi cells and clone 62 hours later, induced CTF secretion, whereas similar CTF secretion was observed in controls. It shows that it cannot be seen.

Although clone 25 secretes interleukin 2, this clone No CTF was secreted from co-culture with irradiated Daudi cells.

Figure 7 shows analysis of CTF by molecular exclusion chromatography. CTF The LK-SN and PMA/Car-3N preparation containing Cefazo7x G10 Also shown is the data of wildflower seeds when chromatographically fractionated at 0sf. It is. In a 6-hour incubation using the assay described in Figure 1. , by triggering IFN-G primed PBM in this column fraction. The presence of CTF was assayed. Other activities were assayed as described elsewhere. Biggest CTF eluted at Mr approximately 55 kd. More minor peaks (approximately 14 kd and above about 150 kd) will vary depending on the induction protocol. Ta. IFN-G cannot be separated from CTF by chromatography. It was.

Detailed description of the invention Abbreviation Act D-Actinomycin D Car-Calcium Ionophore A23187Co-3N-On Control Clear medium CTF = cytotoxic trigger factor FBS-Fetal Bovine Serum GM-C3F = granulocyte-monocyte colony stimulating factor HEPES-N-2-hydroxyethylpiperazine-N-2-ethanesulfani nku acid IL-interleukin IFN” Interferon IFNG” Interferon Gamma LK-5N-Lymphokine-containing supernatant medium LPS=Kyuzobacterium Ribotatou MP　IF　=monocytic procoagelant-inducing factor PBM=peripheral blood cellulose cell PBS-phosphate buffered saline PMA=phorbol myristate acetate 5DS=sodium dodecyl sulfate Mu TNF-A = tumor necrosis factor alpha TNF B = tumor necrosis factor base ■、Introduction The present invention, when primed, produces proteinaceous phosphorus, herein referred to as CTF. After being triggered by the immune system, tumor necrosis factor alpha (TNF- A) It utilizes "effector cells" that can secrete. that Therefore, the effector cells of the present invention are: (1) activated by interferon gamma; (2) Henceforth, the trigger is the bacteria Ribopoly It! (LPS) 1 triggered on contact with a CTF-containing composition, defined as or defined as a large number of cells.

Effector cells are T cells, B cells, etc., as already described. peripheral blood mononuclear (PBM) cell group, including natural killer cells and monocytes. contains cells. The results discussed below indicate that monosites are Primeable and triggerable PBs as discussed above and in more detail below. This is one class of M. Monocytes circulate in the blood and are also fully understood It matures into macrophages that settle in various body tissues by a pathway that has not been To go. Here, monocytes are utilized as exemplary effector cells and Often called “monocyte/macrophage” or “monocyte (macrophage)” ”, indicating the relationship between the two cell types.

To study the effect of IFN-G on the induction of significant cytotoxicity in human monocytes. Recently reported assays for drug-dependent cell-mediated cytotoxicity have been applied and It has been modified and used. Ziegler-H (1984), Journal of National Key Cancer Institute (J, Natl, Cancer In5t,) 7 2, p. 23; and Colotta et al. (1984), Journal ・Of Immunology〇, Immuno1. ) Volume 132, page 936, edited here. This assay has been validated to detect human effector/cell-mediated, e.g. Select mediated in vitro tumor killing activity (cytotoxicity) can be identified and quantified. Hello, T cells, B cells, natural killer (NK) cells , and peripheral blood military nuclear cells (PBM) containing monocytes or purified monocytes only under substantially pyrogen-free conditions (endotoxin-free, LPSo, 0 cytotoxic effector function when released and assayed at was found to be laminar but can be readily induced by endotoxin. Inside In the absence of toxins, monocyte activation-inducible lymph potential for cell-mediated cytotoxicity Other physiological signals, including cancer, can be identified and characterized.

Selectively inhibits the expression of significant monocytic cytotoxicity by primed effector cells The presence and characteristics of a new lynchpin that acts as a second or trigger signal to induce Sexualization is explained by the results described below. (Kornburusu (niorn) blue) et al. 1985 Fed, Proc, Volume 44, 1 699 pages). In this pathway, IFN-G alone is insufficient to induce cytotoxicity. It's a minute.

However, when exposed to IFN-G for a long time, cytotoxicity trigger factor (CTF) and life-threatening of the substantially pure proteinaceous lymphokines of the present invention named Effector cells are primed for the triggering action of the second signal.

CTF alone is not effective in inducing monocytes to express cytotoxicity. It is insufficient. Monosite-like effector primed with IFN-G - PBM containing cells are sensitive to triggers by CTFs.

High concentrations (more than 100 units per milliliter) of CTF alone can inhibit T cells. Trace amounts of endotoxin, PMA, and/or used to stimulate CTF production Cytotoxicity due to the presence of calcium, ionophores or other contaminants It has been observed that inducing The resulting CTF-induced cellular cytotoxicity is With some known biological effects, tumor netarosis factor alpha (TNF- A) Direct effects of monokines (i.e., monocyte/macrophage products) believed to be both attributable and necessary. Old (Old) ( (1985), Science, vol. 230, p. 630.

CT by T lymphocytes as well as by cloned normal T helper cells By describing the immunological production of F. TNF-A dependent tumor killing activity and TNF-A content from human monocytes Physiological pathway for production of internal endotoxin equivalent to external endotoxin in induction of secretion You can draw the outline of Further elucidating the complexity of the MAF controversy and the function of the immune response In addition, substantially pure CTF can be used to improve IF in vivo. A means of inducing TNF-A production in N-G primed monocytes/macrophages provide.

For testing human mononuclear phagocytic cytotoxicity, peripheral blood monocytes are very easily Effector cells that migrate to the site of inflammation when available and successfully recovered represents a group of circulating cells containing Recent studies specific to monocyte-mediated cytotoxicity A rapid assay was developed, i.e., WEH1164 murine mice treated with ActD. Fibrosalcome: 51 (51) released from the target By modifying detection methods such as We analyzed what is required for triggering and triggering.

Contrary to the initial report of this assay, PR11 is present in PR11 derived from normal specimens. monocytes (from humans without disease, inflammation and tumor) - across the board, significantly It exhibited spontaneous cytotoxic activity.

Instead, trace amounts of endotoxin commonly present in culture media, serum, and isolation solutions , is quite sufficient for rapid induction of significant monocyte-mediated cytotoxicity in target cells. It was. Addition of about 0.001 ng/ml of LPS to the culture solution is described was able to stimulate such a response. Endotoxin induces monocytotoxicity Since there is no physiologically sufficient amount of Search and discovery of immunologically relevant second signals or trigger lymphokines Therefore, we revised the analysis system to an extended three-stage analysis system.

n,CTF,Overview IFN acts as the only MAF, i.e. induces monocyte cytotoxicity. The ability of -G has been extensively exploited using both natural and recombinant proteins. 6 Anywhere between 72 hours, whether added all at once, repeatedly, or 0. ．． IFN-G only, whether used at doses from 1 to 10,000 U/l111 Here, we reproducibly target effector cells in PBM to monocyte-mediated cytotoxicity. could not be activated. However, IFN-G (150U in 48 hours) /mI is the second signal that effector cells in PBM trigger cytotoxicity. It has been shown to increase susceptibility to endotoxins by more than 30 times. Ta. Similar results were found by Page et al. (1981) in the journal Reported in Immunology (J, Immunol), volume 126, page 1863. were also obtained for murine macrophages.

It has been shown that the Lymph Cailiin preparation containing crude “MAF” effectively activates monosites. (Kleinerllan et al., 1983) Lymphokine Research (Lymphokine Res,) I S7 page, tab K. Ie inerman et al. (1985) Cancer Lisa Cancer Res, vol. 45, p. 2058, and Talleynerman ( (1984), Journal of Immunology (J, Immunol,) 133@, p. 4), various Lymphin-containing media The ability to induce cytotoxicity against ActD-treated WEHI 164 cells was tested. Ta. Further characterization of two types of media that induce cytotoxicity and their use As with the third type and culture medium, which will be studied in detail, we will discuss it in detail below. discuss.

First, IFN-G, IL2, migration inhibitory factor (MPIF) and monocyte protein Early down (Daudi) cells containing coagelant-inducing factor (MPIF) - activity A culture medium (supernatant medium containing Lymphine; LK-3N) was used. Gregory (Gregory) et al. (1984) Fed Proc 4 3, p. 1748, LK-3N has considerable activity; did not directly induce monocyte-mediated cytotoxicity at an induction time of .

H4bbs et al. (1977) - Science 19 7, p. 279) and Meltzer and his collaborators (Meltzer) Ly+wph 7- (Meltzer) (1981), Ly+wph okines) 'vol. 3, p. 319 and Meltzer et al. (1 982) Fed, Proc, Vol. 41, p. 2198) is a crude The stimulatory effect of continuous administration of MAF was observed. I as the first briming signal The combined effect of FN-G and LK-3N as a second signal was analyzed in this study. .

R, O, production of monosites (Nakagawara et al., (198) 2U Journal of Clinical Investigation (J, Cl1n, I nvest, ) vol. 70, p. 1042 and Gately et al., (1 983) Journal of Immunology (J,ll5+unol) 1 31, p. 2583), enzyme-dependent and -independent intracellular Leishmania donovani - (Leishmania donovani) death (Murra) (1983) Journal of Clinical Investigation U. , Cl1n, Invest, ) vol. 72, p. 1506) and IL-2 receptor (Berman () Ierrmann) et al. (1985) Journal of Experimental Medicine (J, Exp, Med,) Volume 162, 111 reported concentration and time (150 U/m (1,48 ~72 hours) using IFN-G. Under these conditions, the IFN-G Effector cells in PBM incubated with ActD were treated with WEH1162 assay. demonstrated effective monocyte-mediated cytotoxicity in the However, it was limited to when triggered with LK-3N. The observed trigger is L Culture medium from separate cultures of the same cells used to make K-3N was roll supernatant (Co-3N) and mix the medium with the cells, and all media and serum contain no detectable endotoxin (per milliliter). 10 to 30 picograms or less), 0 selections not due to endotoxin contamination of LK-3N In selected preparations, LK-3N failed to induce cytotoxicity in monocytes at 6 hours. induced cytotoxicity in 48 hours. These LK-3Ns include IFN-G Therefore, prolonged exposure to these LK-SNs primes the monocytes. And what's more, you can trigger them. such a choice Since 48 hours was used as the only time for addition of IFN - The rapid effect of non-IFN-G-MAF on G-primed monocytes is It's not obvious and you'll miss it.

Kleinerman and Fiddler ( 1983, Lymphokine Res. Volume 1, page 7 (P activated for 48 hours with concanavalin-A-Sepharose) The MAF-containing preparation made by the method of IFN-G (3 00U/cal1 or more) and the second trigger ring lymph talent in both It contained. Monocytes were treated with this complex MAF preparation for 48 hours or When using long-term cytotoxicity assays (during assays, long-term and large amounts of biological effects ), the priming effect of IFN-G, and the second, more rapid effect of It is not possible to confirm the temporal separation of the trigger effects of Lymph Talent In.

IFN-G was the first molecule identified as MAF and is a monocytosaccharide. To prime effector cells in PBM such as It is often referred to here as MAF-1. Create quickly and a second lymph that is effective on IFN-G-primed monocytes. Sailiin is an IFN-G coupling factor named cytotoxic trigger factor (CTF). It represents. The CTF molecule triggers TFN-G primed cells Therefore, it is consistent with immunological analogues for endotoxins.

Since CTF cannot be replaced with TFN-G, CTF is replaced with IFN-G. (Table 1, results section ■). Furthermore, LK-3N was added to anti-I FN-G When passed through an affinity column containing an excess amount of monoclonal antibody, IFN -G is removed (less than IU/mf remaining by RIA), but CTF activity is , saved. Finally, the relatively high CTF-containing medium is chemically activated. Substantially pure nylon wool-purified normal T cells prepared and produced The CTF Sl product is substantially devoid of IFN-G, a potential CTF source. (IU/m12 or less by RIA).

Since the beginning of these studies, Kramer and his joint research used TF for the induction of cell-mediated cytotoxicity in murine macrophages. Macrophage cytotoxicity-inducing factor-2 (MCIF-2) acting together with N-G reported the existence of a preparation called (Gensa et al. (1984) Immunology (11o1.r+anuno1.) Volume 21, 1267 Hama+ann et al. (1985), European Journal Null of Immunology (Eur, J, Immuno+,) 158.18 In addition to inducing tumor-killing activity, MCIF-2 or one or more of its constituent molecules may In cooperation with TFN-2, intracellular cystosomal ulicida+) activity. Kramer (Kra+nmer) et al. (1985) Journal of Immunology, I mmuno1. ) Volume 135, page 3258.

Cytotoxic activity (Erickson et al. (1982)) Munology (Ce11.Immuno+,) Volume 72, Page 195: Melf-01 (1982) Journal of Immunology (J, I+ muno1. ) Volume 129, page 2802; Melf 7- (Meltzer) et al. (1985), Fed, Proc, Volume 44, 1697a Page; and Kleinerman et al. (1984) Cancer - Research (Cancer Res, ), page 44I4470), antibody-dependent cells Sexual Cytotoxicity [Ralph (1985) Lymph Talent in Research (Ly+1phokine Res,) Volume 3, p. 153), Anti-Leishmania (Leishmania) activity (Hoover et al. (1985)) Fed, Proc, Vol. 44, p. 1697a, and Pacmu Buchmu Ier et al. (1985) Cellular Immunology ( Ce11. In++nuno1. ) Volume 90, p. 242), macrophages herpesvirus replication resistance (Rose et al. (1985)) (Fed, Proc, Vol. 44, p. 1697a), IL-1 Mac Lophage production (Aspen to et al. (1985), Jaana Book of Immunology (J, lm5uno1.) Volume 134, Page 350, and Dinarello et al. (1985) Journal of Ru - Leukocyte Biology (J, LeukocyteBiol,) Volume 37, 6 97a) and unicellular differentiation of leukocytes (Dayton et al., (19 1985), Blood, Vol. 66, p. 583). There are other reports regarding non-IFN-G$B virulence factors. If there was However, the relationship of these factors to CTF has also not been elucidated.

More recently, Lee et al. (1986, Journal op. (J, Im+*uno1.) Volume 134, Page 1322) is IFN-G Identification of human T cell-derived lymphocytes with activity for MAF different from that of and reported the characteristics. This study involved transfection with HTLV-1 virus. All but one model used the 120T cell line.

This report shows that by activating human monocytes, A-315・human, melanoma We show that five cell lines produce culture supernatants containing mediators that kill tumor cells. Ta. The presence of IFN-G in the supernatants of two cell lines exhibiting cytotoxicity Effects on cytotoxicity and monocytic activity of one of these two cell lineages Similar reports have been made regarding the Excess anti-IFN-G monoclonal antibody The reported mixing of these supernatants with monocytes induced by supernatants from these cell lines. There was no marginal effect on the activation of glue killing activity.

In the verification system used here, IFNG and CTF are: 1) 2) does not induce cellular activity in PBM populations lacking sites; 2) isolated Monocytes have the same effect as the same number of monocytes in PBM; 3) their cytotoxicity; The sex factor TNF-A is a product of monocytes, and 4) lI! Microscopic bag of progenitor cells The death observed in most cases is when monocytes attack the abscess. For this reason, it acts on the monocytic subpopulation of PBM cells. appear. Thus, cells of the monocytic lineage undergo CTF-induced mediated While they are considered to be effector cells with cytotoxic effects, they are also responsible for cell activity. The detailed cell subtype or subtype is currently unknown. What theory would you like to combine? At least the monosite can be primed as well as by IFN-G. and the primed monocytes are effectors that mediate cytotoxicity. It is a cell. This means that one or more additional cell types or types may also be present in CTFs. This does not mean that it cannot have induced cytotoxicity.

For example, the disappearance of monocytes from the PBM population is due to natural killer cells and monocytes. It also removes the natural killer cells isolated along with some of the cells.

Conveniently, cells capable of priming CTF-mediated cytotoxicity are referred to as effector cells or model cells. From the heterogeneous mixture called nosite, which constitutes the commonly used non-primed PBM. Primeable cells are called primed effector cells in a broad sense, and are more specialized cells. In such cases, they are called primed monocytes or primed macrophages. Mo Nocytes and macrophages circulate to each other, monocytes circulate while macrophages are distinguished by being descendants of monocytes that are localized in tissues. P.B. Effector cells in M are primed to form cytotoxicity-mediating effector cells In the detailed studies described here, the effector/target cell ratio The number of effector cells stated in is the &8 number of PBM added to the culture medium. , monocytic lineage and not all cells present, i.e., their PBM considers 100% primed for these studies.

Primed effector cells in PBM, such as primed monocytes, are The finding that CTFs mediate cytotoxicity in the absence of PS led to the molecular identification and The mechanism by which cytotoxicity is effective was discovered.

Pace et al. 11981) Journal Sub-Immunology (J, I As discussed by Mmunol, Vol. 126, p. 1863), As detected in the assay described here, the CTF is at its half-maximum - Follows a three-figure dose-response correlation that can be quantified in units. According to kinetic studies, CTF seems to act very quickly and the last 6 hours of the assay of? It was effective even when given simultaneously to target cells during 8 decomposition phases.

Analysis of culture media derived from cloned normal T cells indicates that CTFs are not mechanical products. However, a minor subpopulation of normal T helper clones was induced by alloantigens. Indicates that it is a normal T helper cell product that can be produced after activation. are doing. CTF appears to be a differentiation-dependent product of normal T cell lineages. Also CT F is also a product of certain transformed T cells, as discussed below. .

CTF is well separated from IL-2 and has approximately 55 kilodaltons (kd) (approximately 50 to Sephadex G-100sf (super ・Dispense 7 volumes from Fine). A minor peak of approximately 14 kilodaltons (approximately 9 (about 19 kilodaltons) and minor peaks above about 150 kilodaltons, C It was observed that it exhibited TF activity.

In the assay described above, IL-2 lacked CTF activity. IL-1 is reported to induce cytotoxicity in monocytes (Onozaki i) et al. (1985), Journal of Sub-Immunology (J, [mmunol ) Volume 135, page 314). However, in the above-mentioned assay method, 50U/ mj! IL-1 alpha and beta had no significant effect up to a concentration of Ta. Recombinant colony-activating factor-1 (rC3F-1) also inhibits CTF activity. The approximately 55 kd and approximately 14 kd OCTF fractions showed C3F-1 activity. It was found to lack its activity as well as to lack its activity. In addition, high anger In the L929 cell assay, the 55kd type of PMA/Ca1-5N CTF was also detected. lacked TNF-A or lymphotoxin (TNF-beta) activity.

In this way, CTF is also not a type of cytokin whose characteristics are better known than these. stomach.

Thus, CTF inhibits TNF-A secretion by primed monocytes for 6 hours. Approximate PBMfi degree in normal blood, 2.5X1 per milliliter 0' PBM provides a level of 300 units per millilift dollar or more. It is a cell product. This induction of TNF-A is caused by various T cell commands of the immune response. It shows the physiological role of CTF that is fundamental to the phenomenon and is used in these studies. Mediates cytotoxicity against fibrosarcoma in murine drug-treated mice. in activating primed human effector cells in human PBM. It has ensured its role as a fundamental property.

IFN-G has been reported to enhance the cytotoxic effects of TNF-A on target cells. (Willjan et al. (1983)) , Proceedings of the National Academy of Sciences (P. roc, c, Natl, Acad, 5ci) USA; 80@, 5397 pages; and Stone-Wolff et al. (1984) Jhana. Le Sub Experimental Medicine (J, Exp, Med,) 159 (Vol., p. 828), the apparent priming effect of IFN-G is due to this interaction. It was thought that the While such interactions occur, Effector cell briming is a direct effect of effector cells. exposure to IFN-C; for more than 24 hours and as a second signal. of IFN-G, thereby reducing its effect on target cells. Well-tolerated was not associated with cytotoxicity.

In addition to its ability to prime effector cells, IFN-G is associated with cytotoxicity. TNF-A has been shown to be a necessary reagent to mediate cytotoxicity. was shown. Thus, all induced cytotoxicity is due to the activity of TNF-A. Neutralizing monoclonal antibodies are added to the effluent prior to the lysis step of the assay described here. This problem was resolved by administering it to a primed culture of PBM containing vector cells. . Resolution of this cytotoxicity explains that CTF-mediated effects are TNF-A dependent. are doing.

Although the existence of TNF-AM tumor cells is known, TNFA is Approximately 32 to 64 units per milliliter on average for a wide range of human cell lineages. It has cell-arresting and cytotoxic activities at a concentration of nift (U/m1). oar Old (1985) Science vol. 230, 6 On the other hand, normal fibroblasts are not affected by TNF-A; -A can activate human neutrophil function (Shalab (1985) Journal of Sub-Immunology (J, Immuno 135, p. 2069), which harms the host by causing cachexia. (Torti et al. (1985)) Science (Scie nce) vol. 229, p. 867), and it ) mediates LPS-induced toxicity (Beutler ) et al. (1985) Science vol. 229, p. 869).

To the extent that CTF induces the secretion of TNF-A in vivo Until recently, it has been implicated in host defense 1M and pathogenic sequences in certain disease states.

CTF is TN from IFN-primed effector cells in human PBM. Relatively low dosage as it has selective activity in triggering the secretion of F-A CTF administration at the site of local inflammation and immune response localizes TNF-A-containing factors. Preferably triggers focal secretion to produce significant local or systematic levels of IFNG do.

Often, tumors are overly sensitive to the cytotoxic triggering effects of LPS and their and cannot be distinguished from IFN-G-primed macrophages (Russell Russell) et al. (1977), Journal Sub-Experimental ・Medicine (J, Exp, Med, Vol. 146, p. 1511) of the immune system cells and macrophages (Russell et al. (1980) Conte Comtemp, Top, Imn+uno1. 10 volumes, 143 pages). Thus, clearly physiological accommodation The main response is to prime intratumoral macrophages but not to trigger them. The relatively small dose of CTF at lower concentrations is not the same as the administration of TNF-A itself. other biologically comparable side effects, such as shock, that are observed with Similar to comparable responses, local stimulation of TNF-A-containing tumor IX follicle-killing factor within the tumor It is believed that it confers cytotoxicity to tumor cells through biosynthesis.

■, substantially pure CTF The present invention provides a new substantially pure protein, meaning cytotoxicity triggering factor (CTF). The present invention relates to proteinaceous lymphoid protein, its production method and its use.

CTFs are differentiated, active cells with a T3'''T4'' T8-Ml- phenotype. A proteinaceous lymphoid protein that enables secretion from normal human helper T cells. It is. When expressed from these and other T cells studied, this proteins have an apparent molecular weight of about 4000 to about 150,000 gluton. Sephadex G with a gel matrix that can fractionate proteins According to the value measured by gel exclusion chromatography at 100sf, approximately 5 0 to about 60 kilodaltons (kd), or conveniently about 55 kd or 5 It has an apparent relative molecular weight (Mr) expressed as 525 kd.

As mentioned above, apparent relative molecular weights of about 14±5 kd and about 150 kd or more CTF molecules with The relationship between the molecular species of r55kd and these molecules is currently unknown.

Substantially pure CTF is free of endotoxins. The most common endotoxins The contaminant is lipopolysaccharide, LPS. The fact that it does not contain endotoxins is due to the fact that it does not contain endotoxins. Famous Limulus Amoebosite Lysate or USP Pie You can easily complete 8 laps using the Rosien test method.

In a preferred embodiment, an aqueous composition comprising a triggering amount of substantially pure CTF. more preferably contains less than about 0.0 ing/ml of LPS or other pyrogens. Preferably, such compositions contain less than or equal to about 0.00ing7's+6 of those materials. It must include the following: In another experiment, CTFI units were found to be substantially pure Substantially pure CTF means the presence of 2 nanograms or less of CTF. , most preferably about 300 femtograms of endotoxin (pyrogen) per unit. quality).

Also, substantially pure CTF is substantially free of IFN-G; This does not include IFN-G less than the amount of monosite activation, if it exists. Even if IFN-G is It can be easily removed by using a targeted adsorption column. Possible to exist (non-blamin) Typical IFN-G [is usually about 0.1 unit or more per CTF unit]. Briming amounts of IFN- in substantially pure CTF containing IFN- The presence of G causes the effector cell-containing PBM to mix with a triggering amount of CTF. , and the mixture is incubated for at least 24 hours, more preferably 48 hours. , then labeled Target Act D-treated WEHr 1648E+ cells. Mix and test the solution using the technique described in the dissolution assay described below. The assay can be performed by assaying target cell lysates. trigly The absence of IFN-G indicates the absence of IFN-G.

The existence of direct cytotoxins has been demonstrated by Armstrong et al. (1985) Journal of Sub-National Cancer Institute s-) (J, Natl, Canc, In5t,), vol. 74, p. 1 It can be assayed using a highly sensitive L929 cell assay. Produced in LK-3N The 70kd cytotoxin was chromatinized on Sephadex G-100s4. It can be removed by CTF purification by tography. This site toxic is not present in purified CTF using the PMA/Cal-3N protocol. stomach.

TNF-A is another direct cytotoxic agent not present in virtually pure CTF. It is Shin. TNF, when present, as determined by the L929 cell assay described above. -A was also removed by chromatography using Cefazo7x G-100sf. can be removed. Typically, if discussed in section TV (H) , when an established T cell lineage is used to produce CTFs, TNF-A is not present. do not have.

Substantially pure CTF may also include substantially lymphotoxin (TNF-beta or CTFs obtained from LK-3N often contain no TNF-B). -A or TNF-B, which may contain direct cytotoxins. On the other hand, CTF obtained from PMA/Ca1-3N contains this cytotoxin. do not have. Regardless of its identity, this frequently observed cytotoxin is 14 ．． Both fractions containing CTFs of 55 or 150 kd or more are obtained and used. It can be removed by fractionation using 100sf of Fuazo and Kusu G.

If not identified, the approximately 70kd cytotoxin TNF-A and T NF-B (lymphotoxin) is referred to here as a “direct cytotoxin”. are called all at once. Substantially pure CTF substantially directly removes cytotoxins free of exogenous 70kd cytotoxin, TNF-A and TNF-B. The presence or substantial absence of the above-mentioned L929 cells or Mixing target Act-D-treated WEH1164 cells or The test can be performed using a similar well-known procedure.

Substantially pure CTF is also free of interleukin-2. IL-2 is , was not detected in PM A / Ca I SN, but was detected in LKSN. exists. As seen in Figure 7, the chromatography using Sephadex 0 100s4 By matography, rL-2 can be removed from the water-soluble CTFI proboscis, and it is It can be used to detect the presence of L-2. In addition, IL-2 is transmitted beyond the cell lineage. by the purification method described or by Altman et al., 1984. Co-assigned US patent application registered on April 5th Affinity chromatography as described in number D 97.179 It can be removed by

Cultured in PBM or tanicamycin, the previously described established Substantially non-glycosylated CTFs, such as those obtained from cell lines, also , is captivated here, and also exists in substantially pure form as mentioned above. It can exist.

Preferably, the substantially pure CTF contains less than Both have an activity of about 50,000 units, more preferably milligrams. each containing at least about 100,000 units. most favorable case For each milligram of total protein, substantially pure CTF contains at least about It has an activity of 300,000 units.

Virtually pure CTF is a primed human effector such as monocytes. Effective as a method for causing cells to secrete tumor cell killing factors that induce TNF-A . In this method, for example, primed human monocytes are grown in an aqueous medium. , present in an amount effective to trigger the secretion of tumor cell killing factors, including TNF-A. and a water-soluble composition containing substantially pure CTF. mixed and The aqueous medium containing the cells and supernatant is the in vitro medium in which the contact occurs. ro) provided by cell culture media or treated host blood and lymphatic system. It is an in vivo aqueous medium.

TNF-A from the tumor cell killing factor so secreted is transferred to Sephadex G in vitro on a size fractionation column, such as a column containing 100 sf. tro) As an immunosorbant that passes through the culture supernatant or aims for TNFA. pass it through an affinity column containing the antibody and later elute it from the column. It can be recovered using conventional methods such as

In vitro purification can also be used for the same ffl. In Bi TNF-A produced in vivo is typically It is used in situ (in 5 itu).

The above method can be used both in vitro and in vivo. o) can also be performed.

In vitro aspects of the method include an assay system and This is exemplified in the relationship discussion discussed in detail later.

In typical use, approximately 6X10' to approximately 2X10' units per milliliter culturing the PBM of cells and priming effectors therefrom; and , the triggering amount of substantially pure CTF, e.g., about 3 per milliliter. Approximately 300 units are mixed. The cell culture so prepared was treated with TNFA. When sufficient tumor cell killing factors are secreted from primed trigger cells. maintain for a while. Its typical maintenance time is about 6 hours.

Trigger primed human effects such as monocytes (macrophages) The tumor cell killing factor secreted from the vector cells contains TNF as one component. -A, and likewise other proteinaceous and non-proteinaceous substances. . As discussed in connection with Figure 2, in vitro secretions The presence of TNF- in can be assayed using antibodies directed against TNF-A. . The presence of in vivo secreted TNF-A is associated with IIl tumor cells. This is indicated by the death of the cells.

In vivo embodiments of the above methods typically involve administering a tumor-bearing patient. It was carried out by a person and is described below.

Substantially pure CTF is produced by primed cells such as monocytes (macrophages). ・Human effector cells can be used in vivo and in vitro ( mediates tumor-killing activity (kills tumor cells) in vitro) This is an effective method for inducing people to According to the method, substantially pure CTF water The soluble composition contains tumor netarosis factor-alpha (TNF-A) in an aqueous medium. Trigger the secretion of tumor cell killing factors including and that primed monocytes and a sufficient amount of primed human effector cells to contact the cells.

Tumor cells secreted I! Contact with Igk cell killing factor.

Typical subpopulations of primeable and ultimately primed effectors A variation of the above method using monosites as a Liming is carried out in vivo and using naturally occurring proteins. Provides primed monocytes in addition to primed monocytes (macrophages) do.

Here, circulating effector cells such as monocytes present in the peripheral blood of fools cells are treated parenterally with sufficient IFNG to prime such monocytes. Prime by administering to

The typical effective l FN-G briming amount for such purposes is 11 colors. From about 4 to about 1000 units per milliliter of blood. more preferably , from about 20 to about 200 units per milliliter of patient blood. offer Please note that the definition of the I’1iF-G unit may be different depending on the person. It takes. The above values are from Amgen, Thousand Oaks usandOaks), using recombinant IFN-G” produced by CA. It is from the time.

Generally, IFN-G is a water-soluble, physiologically acceptable, as discussed below. present in the carrier. depending on the patient's specific response to the treatment , one or more administrations may be desirable or necessary.

After the monocyte priming step, the patient receives primed monocytes ( macrophages) for sufficient time and concentration (migration and localization) within the tumor. , primed monocytes (macrophages) that were not concentrated in the tumor were 1. fool It is maintained long enough to disappear from the bloodstream. Typical maintenance times are approximately 5 days (120 hours). The normal lifespan of monocytes in the circulatory system is 3 to 4 days. It is between.

The patient is then administered an effective amount of substantially pure CTF and concentrated contacted with limed monocytes (macrophages) and contains TNF-A. Trigger the secretion of tumor killing factors. Typically, substantially pure CTF is Physiologically acceptable, containing sufficient amounts to trigger primed cells of Administered as an aqueous composition in a carrier. Typical effective trigger amounts are: Will be discussed later.

Again, in a variation of the above method that typically uses monosites, Priming is performed in vitro. Again, that The primed monocytes (macrophages) used are present in Ill tumors. It becomes a form that can be added to something.

Here, human white blood cells, including monocytes, are transferred to the patient to be treated or other comparable human Obtain by leucophoresis of samples from donors. (Leucophoresis is P BM and effector cells, or providing a cell population containing granulocytes) obtained The monosite-containing leucosite was mixed and contacted with a priming amount of INF-G. Prime by touching. An exemplary priming amount for INFC is approximately 1X from about 4 to about 200 units/ml for 10' cells/ml. be. The contact between monocyte-containing leukocytes and IFN-○ lasts for about 36 to about 72 hours. Preferably, when the briming amount of IFN-G is about 20 to about 100 U/mf. The temperature is maintained for approximately 48 hours.

Then in vitro primed monocyte-containing leukocytes Introduced into the patient by intravenous injection. Keep that patient and then trigger the amount of Substantially pure CTF was administered as described above and algal cells were primed monocytogenes. It is left to the killing effect mediated by water.

In another embodiment of the method, the entire method is performed in vitro. o). Here, effector cells exemplified by monocytes are PBM containing PBM was mixed and contacted with a separately stated briming amount of [FN-G. Ru. After that, the primed monocytes (macrophages) will also be discussed separately. Mixing and contacting with an aqueous composition comprising substantially pure CTF as described above. This triggers the secretion of tumor cell killing factors, including INF-A. externally supplied II! Mix and contact i cells with triggered monocytes.

In the above-mentioned in vivo method, the patient's blood is It is recommended to perform a test to determine whether primed monocytes are present in the circulatory system. is preferable. This assay obtains samples containing monosites to be assayed, such as PBM. This can be done by

A triggering amount of a water-soluble CTF-containing composition is mixed with a monosite-containing sample. , the triggering of secretion of TNF-A-containing tumor cell killing factors is described here. The assay was performed by using ActD-treated WEH1164 cells as described in Ru. If circulating primed monocytes are found to be present, the patient The patient's primed monocytes, as described herein, It is preferable not to treat the disease until it is completely gone.

Additionally, prior to in vivo treatment, the patient's monocytes are assayed. , whether the patient's monocytes or other effector cells are primed, and It is desirable to measure whether or not the device is being triggered. This is rare, stupid a person has a genetic abnormality, and their effector cells are primed and triggered. For example, in a mouse system, the mouse C The 3H/HeJ strain does not respond to LPS. The patient's effector cells are described here. Priming and triggering can be tested using a standard three-step assay that has been I can do it.

Primed human monocytes (macrophages) differentiate as already mentioned. This is a mono site. Primed human monosites are not primed Can be distinguished from monocytes (macrophages). Approximately 1 per millirif dollar When in contact with picograms of LPS, 5xlO' plastics per milliliter. Immudo cells were tested for 6 hours in the lysis assay described here. Ii containing TNF-A that kills target cells such as tD-treated WEHI 164 cells Secretes I tumor cell killing factor. Unprimed cells receive more of that factor. secrete smaller amounts, and as a result, under similar conditions, such target tumors i. Non-primed monomers that produce less cytotoxicity to cells. cells are more effective at triggering cytotoxicity than primed monocytes do. It requires 1000 times more LPS.

Thus, this assay can be used to determine whether cells are primed or not. I can do it. Here, three cells containing equal number of effector cells per milliliter A partial sample of section VE, stage! As described in (Priming) Made by III. 525 U/ml in 20 μl of medium! 0) I FN-G , mixed with one subsample (subsample A), while not containing IFN-C, 2 Add 0 μE of medium to other aliquots (aliquots B and C). of the three subsamples Treat each effector cell as described in the priming section.

At the end of the 48 hour incubation period, LPS was added at 1 liter per milliliter. Mix into aliquots A and B in sufficient quantity to give picograms of LPStHr. . At the same time, add LPS to a concentration of 100 nanograms per milliliter. Mix with aliquot C.

The mixture so prepared is subjected to Section VE, Stage II (Regarding) Maintain in incubator for 6 hours as described in .

At the end of the 6 hours, mix the same number of target cells into each aliquot A, B and C and Maintain the mixture as described in Section VE, Stage ■ (Decomposition). Ta. The percent specific release capacity of each aliquot was then determined.

The effector cells utilized in each of the three subspecimens were originally primed. , the percent specific release capacity of each of the three aliquots will be substantially the same. If this If these effectors were not originally primed, their percentage specific release The performance is that subsamples A and C are substantially the same, and subsample B is substantially smaller. Will.

We need to understand whether tumor cells can die by other degradative pathways. but, The degradation and 5IOr-release assays described here demonstrate tumor cell death and appropriate plasma activation. The presence of imdomonocytes is specifically assayed in vitro. This is a simple means that can be used.

The effective primed monosite triggering amount of substantially pure CTF is As is well known in the field, the type and number of tumor cells to be killed. , tumor size (in the case of in vivo degradation), frequency of administration, etc. The inter alia differs depending on the other.

Typically from about 600 to about 60,000 units per kilogram of body weight Essentially pure CTF is administered, with a more typical dose being about 6000 U/kg.

From the perspective of the treated patient's plasma, from about 1 O to about 1000 U per milliliter of plasma; Substantially pure CTF is also typically administered at about 100 U/I11. It is desirable to do so.

Typical in vitro effective amounts are the numbers previously stated. It is a value.

As already mentioned, tumors are primed monocytes (macrophages). Contains. Tumors are also well ductalized, and the ductal system in tumors is It has been found to be relatively permeable to plasma proteins. Saenger (S Enger) et al., Science vol. 219, p. 983, and Dvorak et al. (1984) Cancer Research Re5search) Volume 44, Page 3348.

The presence of a tumor causes monocytes to migrate to the tumor, with only a minimal number of primed cells present. primed macrophages in the tumor compared to other body tissues or fluids that are not present in the tumor. Concentrate the medium. The relatively high permeability of the tumor pathological stem is due to the Orally administered CTF was applied to tumors with the highest concentration of primed monocytes. to concentrate. Thus, administration of CTF by injection to patients with tumors is a monotherapy. CTF is concentrated at the site of highest concentration of cells (macrophages).

In this way, substantially pure CTF can be delivered into the patient in a physiologically acceptable manner. injecting a composition comprising an effective amount of CTF into an aqueous composition comprising a carrier; As a result, CTFs are transported to the tumor cells by body fluids such as plasma or lymph. in vivo by parenteral introduction, such as by It can be brought into contact with limed monocytes (macrophages). ! ! :, Typical parenteral introductions into patients include intramuscular, intraperitoneal, intravenous, intratumoral and similar Includes injections and infusions.

CTF is present within the wound and virtually absent elsewhere in humans without inflammatory conditions. activated or primed monocytes (macrophages) that are not present in Note again that it acts only on effector cells. In addition, Makurov The archetype is relatively immobile and its precursor, unprimed model, As a result of these facts, the actual Systematic in vivo administration of qualitatively pure CTF improves the ability of tumor cells to Local killing, ie, substantially killing only the tumor site.

A more detailed analysis of the environment in which tumor cells die shows that in vivo (jnvt vo) mode, primed monocytes (macro) triggered in the patient. phages) are essentially primed within the patient's tumor. Only clophages.

Furthermore, tumor cells contacted with a TNFA-containing tumor killing factor are Monocytes are the cells of the tumor that are triggered.

If the method is carried out in vitro, human monocytos was successfully primed as described here using IFN-G. Ru. Similarly, contact between primed monocytes (macrophages) and CTFs and , targets like ActD-treated WEH1164 cells! I! Tumor cells and TNF-A-containing Contact with existing primed monocyte (macrophage) secretions is not discussed here. Well maintained as shown. Monocyte (macrophage) triggerin Within approximately 10 hours after initiation of testing, in vitro (in vHro) systems of external origin Target L during timing! tumor cells and target tumor cells in the mixture. The limed monocyte (macrophage) ratio ranges from about 0.6=1 to about 20:1. and more preferably about 10:1.

Substantially pure CTF of the invention, either in glycosylated or non-glycosylated form. can also be obtained by freeze-drying a suitable aqueous composition (solution or dispersion). can.

Water-soluble physiologically acceptable carriers are well known in the art.

All carriers must be sterile, substantially pyrogen-free (e.g., endotoxin-free). Contains water (without) and also contains Limulus amoebosite lysate (Limul US amoebocyte 1ysate) assay method or USP pyrogen Test (Pyrogen Te5t) (The United Stake Far Makopeia (The United States Pharmacopeia) 11kJ4.863 (1965)). ing/all or less, preferably 0.00 ing/ca1. including: It is.

Briefly, following the USP Virogen Test, the water solution containing the substance to be determined The sexual composition was administered at a rate of 10 milliliters per kilogram of body weight within 2 minutes. Three rabbits are injected intravenously. Body temperature is measured 1.2 and 3 hours after injection.

If there are no rabbits with a body temperature of 0.6°C or higher than normal, and if there are three If the total increase in body temperature does not exceed 1.4°C, the test substance is fever @! absence of quality Meet the requirements.

Useful carriers typically include those that are free of substances other than CTF and water, or that are A buffer such as sodium phosphate, physiological saline, or phosphorous at a physiological pH value An aqueous solution containing both, such as W-buffered saline. In addition, water-soluble carriers salts such as sodium or potassium chloride, as well as one or more buffer salts, sugars such as glucose, mandol and dextrose, serum albumin Proteins and other solutes may also be included. These latter killers include Ringer's injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactate exemplified by Ringer's injection.

The water-soluble CTF-containing compositions useful herein can be added to the method of the invention at once or More specifically, it is administered several times over a period of weeks to months of treatment. be done.

Substantially pure CTF can be used as a composition of the present invention or as a composition on its own. It can be packaged in dry form to be dissolved before use. packaged as such For each package, the CTF is a predetermined value calculated to produce the desired effect. Physically suitable as a unit dose for administration, containing a given amount of Lymphin. It has a separate unit administration type. Examples of suitable unit dosage forms include powder Includes sachets, pellets, ampoules, vials and the like.

■Results A. Basic certification system For the assay of monocyte-mediated cell-mediated cytotoxicity, previously described and human Actinomycin, confirmed to be specific for cell-mediated cytotoxicity (Act) D-treated WEH1164 mouse fibrosarcoma cells "Cr release" An assay method was applied (Ziegler-Heitb (1984) Journal Sub-National Cancer Institute Incubate (J, Natl, Cancer In5t,) 721! 23 pages and Colotta et al. (1984), Journal Sub-Immuno. Logi (J, Immunol, Vol. 132, p. 936), in the 6-hour test It was modified and used.

This target cell has natural killer (NK) 13 cells, polymorphonuclear nuclei. traphilus (PMN) and damage mediated by human T lymphocytes. showed resistance to

from healthy, normal volunteers (e.g., with known diseases, inflammatory conditions, and tumors). If PBM isolated without scrupulus endotoxin has this target Even if there is a naturally occurring cytotoxic effector function for Contrary to what Oe's reported research results indicate, it reiterates that there are very few It was found that this is actually shown. However, Escherichia coli (E, coli) 0111:84 Trace amounts of LPS from the strain rapidly induced cellular cytotoxic functions. (For example, Figure 1 , see panel B) Endotoxin is a physiologically irrelevant signal in a normal individual and typically In immunologically mediated damage, the observed monocyte-dependent cell-mediated The assay does not require monocytotoxicity because it is not present at the concentration necessary to induce monocyte toxicity. exert similar biological effects on cells, especially those that trigger the induction of cell-mediated cytotoxicity. Looking for new lymph talent to act as a “second signal” or as a “second signal” It was modified to be longer and in a three-stage format (Figure 1, panel A).

A medium rich in lymph talent for the initial exploration of triggering and lymph talent. A unidirectional, mixed allogeneic leukocyte reaction was used to generate. normal human effe PBM containing vector cells and irradiated Daudi B lymphoblast cells from 1=3 to 1= By co-cultivating for 18-24 hours at a ratio of was generated. PBM and irradiated dows as control supernatant (CO-SN). Cells were cultured separately and their supernatant medium was mixed at a ratio of 1:3 to 1:5 (v /v).

Preparations of LK-5N were prepared with varying amounts of IFNG, IL2 and monocytopropylene. Coagelant/gK factor CMPIF) (Gregory et al. (19 (1984) Fed, Proc, Vol. 43, p. 1748). Normal mode Nocyte cytotoxicity was observed in PBM mixed and maintained with LK-3N for 6 hours. It is missing or close to its limit.

Similarly, recombinant IF N G (r IFN-G) was also completely and reproducibly monocytosed within 6 hours. Some research studies (see Figure 3B) are unable to induce cell-mediated cytotoxicity. Research shows that IFN-G, which functions as “MAF-1”, activates monocytes. (Nakagawara (Nakagawara)). wara) et al. (1982), Journal Sub-Clinical Investigation tion (J, Cl1n.

Invest, ) vol. 70, p. 1042), Gately et al. (1 983) Journal Sub-Immunology D, Immunol, ) 131 Vol. 2583, Ierrman et al. (1985) Journal. Sub-Experimental Medicine (J, Exp, Med,) Volume 162, 11 (p. 11), in a three-step assay using PBM with or without various reagents. Culture and maintenance (incubation) took place for 48-96 hours. comprehensive screening Figure 1 shows the representative results of this research.

The principle finding, observed reproducibly using PBM from eight donors, is that IF When mixed with and maintained in contact with N-G primed cells, LK-3N significantly (Figure 1, panel C). PBM at various concentrations induces toxicity (Figure 1, panel C). When cultured in medium for 48 hours without the first signal given by N-G, the standard Addition of LK-3N as a second signal 6 hours before addition of target cells resulted in significant cytotoxicity. Does not elicit a sexual response.

However, when PBM When cultured for 8 hours, the addition of LK-3N as a second signal Significant cytotoxicity was induced against EH+164 target cells. If from outside IF, 'J-G is not added, and PBM is mixed with LK-3N for 48 hours. When cultured (including IFN-G50-300 U/vsl! from inside) , typically monocytic cytotoxicity is observed.

On the other hand, under any conditions, the control medium supernatant (Co-3N) was significantly It is not possible to induce significant cytotoxicity (Figure 1, panel D). These effects are I Since the addition of FN-G alone cannot induce a cytotoxic response, the first and act as a second signal to the IPNG present in the LK-3Nill product. (Figure 1, panel B).

Lymph protein produced by nylon wool enriched human T cells - Rich media was also tested as a second signal lymph input source. main concentration phorbol myristate acetate) (PMA) and calcium io Nofuor A231B? (Car) (PMA/Cal-3N) for 3 hours. After 48 hours of culture, the IFN-G contained in the medium was IU /mj' (Figure 1, panel E).

Induction of cytotoxicity followed by a series of PMA/Ca1-3N induction protocols. The lymph concentration required for the LK-3N is significantly greater than that in the won.

Cefadefux G-100sf fraction as approximately 55 kd PMA/Car-3N as a second signal. When used, effector cell-containing human PBM with IFN-C for 48 hours priming significantly enhanced its effector function. I'm researching further However, some cytotoxicity was present without priming, which may be due to inadvertent It is thought that this is due to contaminants that are intentionally present.

First, these studies showed that PBM has an autologous effect on ActD-treated WEH1164 cells. It has been shown to lack spontaneous cytotoxicity.

Second, endotoxin is one inducer of this latent activity. Thirdly, IFN- G alone is not sufficient to induce this activity. Fourth, inducible lymph ability Force-in, i.e., CTF, is a chemically activated (PMA/CCa1-3N)P B and T lymphocytes as well as immunologically (LK-3N) are released into the culture medium.

Thus, due to the induction of significant cytotoxicity, IFN-C; (MAF-■) It acts as a liming lymph talent, and the CTF acts as a trigger ring. This explanation is supported by the research results that follow. ing.

B. Effector cell specificity ActD-treated WEHIL64 cells are killed by NK, T lymphocytes and PMNs. They are monocyte cytotoxic as they are known to be resistant to annihilation. useful as a target against

(Ziegler-Heitbrock, etc.) (1984) Journal of Sub-Nal Cancer Institute (J, Natl, Cancer In5t,) Volume 72, page 23; Colo 7 Colotta et al. (1984) Journal of Sub-Immunology (J , Immunol, ) 132@, p. 936), endotoxins involved in cell fractionation (e.g. (LPS) purified products due to the risk of contamination and the possibility of multiple interacting cell populations. Unfractionated PBM is used as the effector rather than the site.

However, it is possible to mix external lymph talent with PBM used as an effector. Therefore, other types of lymphoid activated killer cells may be induced. (Grins et al. (1982), Journal op Exverime Mental Medicine (J, Exp, Med,) vol. 155, p. 1823, Warren ( (1984) Journal Sub-Immunology (J, Imn) +uno1. ) Volume 132, page 2888, Svedersky ) et al. (1985) Journal sub im, nology (J, In+muno 1. ) Volume 134, p. 1604, Yang et al. (1985), J. Null Sub Immunology (J, io+IIuno1.) Volume 134, 391 2 pages). Thus, the specificity of this method for monosite-mediated killing was reevaluated. It was.

Specificity of TNF-A cytotoxicity in L929 cell assay for TNFA monoclonal antibody (TNFD) (Arwstr. (1985) Journal Sub-National Cancer Institute Institute (J, Natl.

Canc, In5t, ) Volume 74, Page 1), ActD-treated WEHI164 cells The IFN- counteracted the cytotoxicity induced by CTF in limed PBM (Fig. 2, Panel A). In the absence of effector cells, TNFA alone and the presence of IFN-G Bottom, cytotoxicity was induced to the same degree as that induced by CTF. (Figure 2, panel B) In this way, TNF-A can be combined with IFN-G to induce PBM by cTF. is required to mediate the cytotoxicity induced in the cell.

The fact that TNF-A is a monocytic/macrophage product (old (O) ld) (1985), Science, vol. 230, p. 630 ) further demonstrated that the ActD-treated WEH1164 cell assay is a monocyte-mediated This provided confirmation that the cytotoxicity was specific. As a result, that INF-G Prime The CTF-activated effector cells are at least monocytes and/or macrophages. Defined as clophage. However, this definition excludes the inclusion of other cells It's not a thing.

C. Temporal and quantitative requirements for IFN-G briming - Throughout, IFN- PBM pretreated with G ) are observed to be more sensitive (or primed) to the effect. I FN-G briming itself acts as an activator, but it also acts as a cytotoxin. or homologously activate PBM in irradiated Daudi cells that do not produce CTFs. Detecting lower levels of CTF present in LK-3N produced by required for release.

A fixed amount of LKSN was added at various incubation times during the triggering phase of the assay. Mixed with effector cell-containing PBM primed with 150U/■l IFN-G. When combined, 24 hours are required for the briming effect to become evident (Figure 3, Panel A).

Tal I F N -G dosage, ('b) during Snurge I priming of the mixture Response to triggering signals using extensive checkerboard analysis between Effective briming for is evident at 36 hours and complete by 48 hours. administration of IFN-G may promote or heighten it. It didn't. If IF is present for up to 96 hours in culture, briming is reduced. As an exception, after triggering LK-3N and immediately before target cell mixing, I Mixing FN-G showed various mild increases in cytotoxicity. This is - piercing LK-3 used as a second signal to trigger PBM. Nm products contain cytotoxic factors such as TNF-A or lymphotoxin (TNF-A). -B) was also observed. Thus, for all preparations of CTF, Analyze the presence of direct cytotoxins and virtually eliminate direct cytotoxins. Use only preparations that do not contain cefazo, Fractionated with f to produce a substantially pure, concentrated, substantially direct cytotoxin-free product. It is necessary to recover pure CTF.

The effect of IFN-G may not simply be due to a cytotoxic synergy with the induced TNFA. stomach. Such a synergistic effect may be responsible for the apparent increase in cytotoxicity produced by rFN-G. (Williamson et al., (19) 1983), Proceedings in National Academy Sub-Saie (Proc, Natl.

Ac1d, Sci, ) U S A vol. 80, p. 5397; and Stone War Stone-Wolff et al. (1984), Journal op. Suberimental Medicine (J, Exp, Med, Volume 159, Page 828) , cannot explain the time required for IFNG to prime to maximum effect, this is , through receptor-mediated consumption of IFN-G by cells present in the PBM. This occurs after 48 hours, a time of relative depletion from the medium. In support of this explanation Therefore, IFNG can induce concentric time to induce other macrophage activation effects. Nakagawara (48 to 72 hours) is reported to be necessary. (1982) Journal Sub-Clinical Investigation tion (J, Cl1n, Invest,) vol. 70, p. 1042, Gately (Gately) et al. (1983) Journal of Sub-Immunology (J, I n+muno1. ) 131 @, p. 2583; Murray et al., ( 1983) Journal of Sub-Clinical Investigation (J, Cl. 1n, Invest, ) vol. 72, p. 1506 and Herrman. ) et al. (1985) Journal of Sub-Experimental Medicine (J, Exp, Mon ed,) Volume 162, Page 1111.

Various doses of PBM were administered to determine the dose of IFNC required for briming. amount of IFN-G for 48 hours, then an effective amount of CTF was added. IFN-G was triggered for 6 hours using LK-SN (Figure 3, panel B). Varying the dose value for the primary effect between 4 and 20 UZIlll and also Imaging is 20-100U/mj! It was the best when. Natural IFN-G (nl FN-G) and &II replacement I FN G (rl FNG) are PBM showed no significant difference in the ability to prime.

As shown in Figure 3, Panel B, doses of up to 5000 U/ml of IFN The following is the second signal rifling power input, namely CTF, of the trigger ring. In its absence, it was not possible to directly induce cytotoxicity. This applies to all LK-3 This is approximately 100 times greater than the IFN-G concentration found in Nfi products.

In the I FN-G briming checkboard/analysis, the maximum at 48 hours Dosage higher than the dose may successfully promote its priming effect or I didn't get excited. As a result, in all studies of CTF requirements, 150 U/mj for 48 hours to prime vector cells! IFN-G and reaction was used.

D, MAF-n) Temporal and quantitative requirements for reggregation IFN-G prime Mixing of OCT FB externally into PBM containing effector cells and its generation Maintaining a contact with It rapidly induces sexual activity (Figure 4).

Substantially pure CTF of LK-3N or 55kd fraction of PMA/Ca1-3N The triggering interval was reduced from 6 to 0 hours. A series of five training sessions In our study, the triggering of monocyte-induced cytotoxicity by LK-3N was After some time, it increased to the highest value. Consequently, the CTF reaction time can be calculated using a systematic analysis. 6 hours for the cytotoxic effector, 6 hours as the previous triggering time for 1 hour. Time was adopted.

To test the required amount of CTF, during the 6-hour triggering phase, IF N-G primed PBM effector was mixed with various concentrations of LK-3N or PMA. /Ca1-3N was mixed with substantially pure CTF of the 5 skd fraction. produced The cytotoxicity layer has an 8-figure correlation with the added CTF dose, with the highest activity. reached a plateau (Figure 5).

Under these conditions, the MAFnl unit has 104 ActD-treated WEH116 4 target cells to target cell effector ratio (E:T) of 50:1. in IFN-G primed PBM in 6 hours. Defined as the amount of CTF that induces cytotoxicity of xime (150 U/ml in 48 hours) Ta. In these examples, the LK-SN preparation typically used was 44CTF Contains U/ea1 and is commonly used P M A / Ca I S N contains 100 CTF U/mJ. 4 hours after secretion, C-10/MJ2 cells Cell serum-free medium (discussed in Section IV (H)) contains approximately 2000 U/+ ++1 or 40.000 U per ■ protein, and further purification results in total It gave an activity of about 300,000 U per protein.

E, Production of MAF-II by cloned human T lymphocytes, normal alloantigen responsiveness. H) Activation of T lymphocytes with irradiated Daudi cells and cloning using conventional methods It was made in vitro. Gregory (1985), Journal of Clinical Investigation (J , Cl1n.

Invest, Volume 76, Page 2440.

51 normal T cell clones were selected and C The ability to produce TF was analyzed. The mulberry clone has a suitable proportion of irradiation. Incubate for 4 to 72 hours in the presence or absence of cells (depending on the clone). 2 to 6 Dauzi cells). Harvest the medium and CTFs were analyzed using IFNG primed PBM as a vector.

As shown in Figure 6 for two representative clones, CTF was It was not observed as a constitutive T cell product in vitro). main street Is homologous activation as a phenotype? 3” T4°T8-?11- CTF was produced using Parklawn 62.

On the other hand, clone 25 was under the same expression and it also produced IL-2. However, no CTF was produced.

T3''　T4''　T8-　by normal Ti$EI cell clones other than Ml- phenotype No CTF secretion was observed.

These results clearly allocate CTF biosynthesis to normal T helper cells. , implying the necessity for T receptors to be involved in the control of its biosynthesis. However, CTF production by other cells, such as yet unidentified cells. This does not completely exclude the possibility of In addition, the Belper 7 phenotype is similar to CT Although it appears necessary to determine those T cells capable of secreting F. I don't think that's a sufficient standard.

Physicochemical characteristics of F, CTF CTF is a single independent molecule rather than the combinatorial effect of a mixture of molecules. The clearest evidence of this is Sephadex G-100 Super Fine. (sf) by sciss fractionation by molecular exclusion chromatography.

The greatest CTF activity is under non-dissociative conditions with an apparent relative fraction of approximately 55 kd. It eluted with good reproducibility as a bright peak with molecular weight (Figure 7). in some preparations whereas one or two additional peaks with marginally detectable activity kd and an apparent Mr of 150 kd or more. Such a conclusion The result is that 55kdCTF may exist in smaller or larger molecular weight forms. Inducing cytotoxicity of target tumor cells by aggregate or primed PBM One or two different additional molecules may also be secreted from Tt+EI cells that can It shows.

CTF activity was recovered well separated from IL-2, and cytotoxin In some cases, it was found in LK-3N preparations. The 55kdCTF positive The tabular peak lacks activity in the migration inhibition factor (MIF) assay and is for Ronnie activating factor-1 (C5F-1) by radioimmunoassay. negative and granule cell-macrophage by bioassay. It was also negative for colony activating factor (CM-C5F).

When IPNG is present in LK-5N or PMA/Cal-3N, 55 kdC Although the TF positive peak co-elutes with IPNG, a minor 150k The d and 14 kd peaks contain no detectable IFN-G, and furthermore, IF Even when NG was reduced from 55 to less than lU/rail, CTF activity remained can be removed from LK-3N preparations by an N-G immunoaffinity column. I can not do such a thing.

In addition, PMA/Cal- prepared from nylon wool-purified normal human TE3 cells 3N lacks IFNG, IL-2 and cytotoxin, while it A major peak of about 55 kd and one minor CTF peak of over 150 kd. It was found to contain. In assays for TNFA or TNF-B, PMA- Cal-3N-derived 55kdCTF has direct cytotoxic activity against L929. It was missing. Armstrong et al. (1985) Jar Natl Sub National Cancer Institute) (J, Natl, C anc, In5t,) Volume 74, 1. In this way, CTF can be used for MIF, C3F-1 and and GM-C3F, IFN-G, IL-2, TNFA% TNF-B or It is different from other direct cytotoxins.

G, other well-characterized sidekins do not function as CTFs.

Standard three-step test using IFN-G primed effector in PBM A variety of well-characterized cytokines can function as CTFs. I tried it. The results of this test are shown in Table I below. A summary of the differences with the same type is shown in Table ■ following Table 1.

Table■ Absence of activity of well-characterized sidekins in basic CTF assay 1 CTF (LK SN55kd N, D,' 31.7±18.7G 100s f fraction) nl FN-G 10 U10+lO, 5±6.1100 6.4±3.2 1.000 0.0±7.9 10.000 2.0 = 3.1 rl FN G 10 [1/111-1.6±4.7100 4.2±4.2 1.000 2.7±4.7 10.000 -2.1±5.9 n I L 2 1.6 U/mj! 4.2 = 4.2 Contamination by IL-1 6 ．． 25 -4.0±5.1 and Limulus test method 25 18.6 = 279 positive for endotoxin due to positive number 1 CTF (LK SN) 111 Ll/llj! 64.7±7.4n I L 1 (beta) 1.6 U/ll11 10.9±6.83.116. 9±5.6 6.3 11.8=2.2 12.5 19.9±1.6 25 16.2±3.4 rGM-C3F 7.8 ng/m7! 32.3 == 11.531 22.8 ±6.6 125 28.3! 14.5 500 30.8 = 8.4 rl L-25U/mn -9,9±4.550 -1.4:3.9 500 19, Oi 3.0 Stop calibration CTF (LK-3N55kd N, D, 30.0:4.0G 100sf image minutes) CTF (PMA/Cal-3N) 57 U/+off 63.2 Sat 3.6n I L 1 (beta) 0.5 Lf/+oj! -2, 1.21 - 2.8 Sat 0.9 5 o, s=4. t lo　6.1=2.2 50 7.2±5.0 stopping ground CTF (LK-3N) 44 U/van 30.4±6.6r I L - 1 (alpha) I U/sl 10.1 Sat 2.03 10.3±1.6 10 10.3±3.5 30 12.9±5.5 r　I　　-1(beta)　I　U/nai! 8.7±1.33 10. 5±0.3 10 7.7±4.0 30　19, S±7.9 Stop calibration CTF (LK-3N) N, D, 28.3±3.3rCSF-11U/m6 13.5±2.510 9.5±3.9 100 11.8: 1.9 1.000 32.1±4.7 1. Cytotoxicity in “Basic Assays” is described in “Materials and Methods” Section V (E ). Cytokin used for comparison is Section V Derived from those listed in.

2. Concentration is expressed in units per milliliter (U/m1) or nanoliters per milliliter. It is given in ng/+sI.

3. As defined herein, cytotoxicity is reported as percent specific release capacity ± standard deviation. be notified.

4, N, D, = not measured.

Table■ Summary of differences between CTF and other well-characterized sidekins Cytokin interaction with CTF Even if the level in IFN-(, (a)LK-SN is 100 times or more , unable to induce cytotoxicity.

(bl) Monoclonal antibody immunoaffinity without loss of CT F activity. It can be removed from LK-3N using a column.

It does not exist in PMA/Car-3N including tel CT F.

IL-2 (at rock-bottom levels, such as that seen in alLK-3N) is generally cytotoxic. I can't induce it.

(In bl Sephadenx G 100sf, it is separated from CTF and eluted.

It does not exist in PMA/Ca1-3N containing tel CT F.

IL-1 (al LK-SN level more than 10 times higher) However, it cannot induce cytotoxicity.

G M - CS F (al L K - S N) However, it cannot induce cytotoxicity.

World) LK 55kd Sephadefex G-100sf fraction containing SN and CTF does not exist.

TN F-A (55kd Sephadefx G-100 including al CT F It is not present in the sf fraction.

(It does not exist in PMA/CaI-3N containing bl CT F.

T　N　F　-B　(55kd Sephadefx G-100 including al CT　F It is not present in the sf fraction.

CS　F　-1tel　CT　Has no F activity.

(It is not present in the 55 kd Sephadefex G-100sf fraction containing bl CT F. do not have.

MIF falcTF is present in the 55kd Sephadenx G-100Sr fraction. Doesn't exist.

The trials in the table above include IFN-G, IL-2, IL-1-alpha and beta, GM -C3F%C3F-1, TNF-A and TNF-B are included in any CTF positive preparation. It is confirmed that the amount present in the substance does not replace CTF or function as CTF. It shows.

Thus, all of the tested sidekins were observed in all CTF-prepared proboscises created in this study. It was concluded that this was not related to the expected CTFT properties. This conclusion is independent It is consistent with CTF as a molecule.

H, Production of CTFs by transformed T cells Previously in Section Iv(E) The work described in 2012 is based on normal human T helper cells, i.e. those derived from healthy humans. It is associated with CTFT production in T helper cells. described in this section The work involved developing CTFT cells derived from T cells transformed by HTLV-1. It is related to

The particular HTLV-1) transform cell line used was named CIO/MJ2. and National Institute Hob Health, Bethesda, MD. Stored at the National Institute of Health were used and were available to qualified researchers. This cell lineage is Developed by Dr. Robert Ga1lo and his collaborators Science, by Popovic et al. (1983) (Science), Volume 219, Page 856.

In addition to the CIO/MJ2 cell line, other transformed or malignant T cells The cell lineage can also secrete CTF. Examples of these cell lineages include 0 M3104A, National Institutes Hob General Med. Cal Sciences Human Genetics, Re-Mutant Cell Revo The National In5titutis of Gene ral Medical Sciences Human Genetic Mu Lymphoblast fine available from tant Ce1l Repository) cell lineage; and HuT originally obtained from the peripheral blood of patients with Mycosis funcoides. 102 (ATCCTlB162), the C10/MJ used here 2 cells in medium without protein, approximately 0.5-2X10' cells/l111 Proliferates to an optimum of 4 degrees.

The medium used is the “medium” described in Section V(A) excluding FBS. Ru.

These CIO/MJ2 cells were incubated at 1 micrograss/II for 4-8 hours. in the medium in the presence of the glycosylation inhibitor tunicamycin present at a concentration of one or more secretes CTF. In addition, contrary to normal helper T cells, CIO/MJ2m The cells are exposed to external activation as well as by PMA/Cal or irradiated Dauzi cells. Without sexualization, they are likely to secrete substantially continuously impure CTF. It is a constitutive CTFT producer.

Substantially pure CTF was obtained from cultures of CIO/MJ2 cells as follows. It will be done. The cells were incubated at 0.5-4X10 in protein-free medium for 3-8 hours. ' cells/aal. The culture medium supernatant is usually used Separate from cells by low speed centrifugation. The supernatant from the centrifugation was further heated at 4°C for 1.5 hours. , 100.000 g to remove cell debris and HTLV-I particles.

The clear liquid is then passed through a 25 nanometer bore filter and the remaining Remove virus and cellular debris. The activity of CTF prepared in this way is , approximately 40,000 units per milligram of protein.

The CTFT product was dialyzed against PBS, pH 7.2, using an Amicon ultrafiltration device and Concentrate by ultrafiltration using a YM-10 filter, and further add cefazo Fractionation was performed by gel exclusion chromatography using 7xG-IQOsf. main Essentially, a substantially pure CTF protein with a value of approximately 55 kdOMr was obtained as previously described. As described above, it has a relative molecular weight of about 14 kd and about 150 kd or more and CTFT properties. Two minor substantially pure proteins were recovered as well. Approximately 55kd The activity of substantially pure CTF with a 1r value of , approximately 300,000 units.

■Materials and methods A. Culture material All reagents are virtually endotoxin-free (less than 0.0 Ing/ml). prepared in endotoxin-free water (Deerfield, IL). ieM), Travenol Laboratories Ficol+ 400 (St., MO) Louis, St., Sigma Chemical l Co, )) is dissolved in such water, and the density is 1.074 per milliliter. Hypaq%'e (50% diadrisol) Winslow 7 Lab, Menlo Park, CA Winthrop Laboratories was added.

RPMI 1640, which is known to be free of endotoxins (Grand, NY) Island (Grand l5land), GIBCO (GIBCO) or MD Walkersville, State ttaker) M, A, from Biochemicals The selected lofts (obtained) were used in all studies.

HEPES (4-(2-hydroxyethyl)-1-piperazine ethyl Sulfonic acid; CA, La Jolla, heh! J song Diagnostics (Behring Diagnostics), Calva CALBIOCHEM Bio Chemica ls)) in endotoxin-free water to a concentration of 1.5 molar-CM). Then, using NaDH, RPM11640 was adjusted to 25 millimolar (mM). At this time, the pH was adjusted to 7.30. “Medium” is Mililiat 50 micrograms of gentamicin (Kenilworth, NJ) per bottle Schering Corp. , ), which contains 251 Hebes, and which does not contain substantially any of the selected 10% Fetal Bovine Serum (Santa A, CA) na% Irvine Scientific 7ri (Irvine 5cientifi) c), KS, Lenexa, K, C, Biochemicals (B iolo (icals) and Logan, IT obtained from Sterile System+s, Inc.) I made up for it. It is RPM11640.

Phosphate buffered saline (PBS) (NaCl, 8 grams per rifdle, 10.2 g/12SNa) IgPOa 0.2 g/Il, NaJPOn -7HzO 2.16g/l) was made with pyrogen-free water and its pH was 7.20. . Trypsin-EDTA was obtained from Irvine Scientific (Irvine 5). scientific).

96-well U-bottom tissue culture plate (#3799) and 75+U2 flask are available from Cos. Coster (Cambridge, MA) Obtained from. 50ml polypropylene tubing is manufactured by Corning Grain Cornig GlassWorks (Cornig, NY) Obtained from Corning.

B. Drugs Heparin, 500 units per milliliter (N, H) Cherry Hill), Elkins-3tnn Co.) ; actinomycin D (Act D) and phenol derived from E. coli 0111:B4 Extracted lipopolysaccharide (LPS) is produced by Calbiochem Biochemicals (CAL). B10 CHEM Biochemicals). chromic acid 5ICr and Aquasol as sodium are available from Das, MA. Danvers, purchased from Amicon Corp. I got it. Sephadex G-100sf is manufactured by Piscataway, NJ. cataway), Pharmacia Fine Chemicals (Pharmacia Fine Chemicals) Fire Chemicals). IMRX Interferon- Gamma RIA is located in Malvern, PA and Centcole (CE). Obtained from NTOCOR).

Preparation of C1 cells Peripheral blood monocytes (PBM) were given pevaline at a final concentration of 5 U/■l. Healthy (normal) volunteers (known tumor or other monocytic brimming) was prepared by collecting venous blood from patients (without the condition). 12 of that blood Centrifugation was performed for 25 minutes at 0xg and the platelet-rich blood was removed. The blood is then cultured. The volume was returned to its original volume in the ground, and a second centrifugation was performed to remove platelets.

Following this, Ficoll-Hypaqve The effector cell-containing PBM is isolated using FBS-free medium. Washed three times with These cells showed 98% by trypan blue exclusion test. More than 1 cent alive, measured by esterase staining, 15-20 It contained % monosites. In some cases, monocytic deleted T EI cells by Falkoff et al. (1982), Journal ・Sub-immunological methods (J, Immunol.

Methods) Volume 50, page 39, nylon wool fibers Refined with rum. Unless otherwise noted above, all media are 10% This supplements the FBS.

D, staining of target cells Mycoplasma-free WEHI 164 murine fibrosarcoma cells ( National Cancer Institute Ronald Herbe of In5titude ra + an) provided by Dr. 10% FBS without selected endotoxins at 37°C in an atmosphere of Supplemented endotoxin-free I? Cultured in PM11640 medium. target cells, Two days before cultivation, the cells were passaged using trypsin-EDTA. On the day of the study, cells were harvested. The cells were removed from the culture vessel while being gently shaken using pushin-EDTA. sign 5XIO'WEH1164 cells were cultured prior to Falkoff et al. (1982), Journal of Sub-Immunological Methods (,T,Im Munol,! 1 methods) As reported in Volume 50, page 39, 50m j! In a tube, 1 μg, /+a7! in 30 ml of medium. Using ActD, 3 o'clock pre-treated for 30 minutes, then pelleted by centrifugation at 120 x g for 5 minutes, and ．． All but 5 ml of medium was removed.

200 microcuries of Cr (200-900Cj/g) in normal saline solution ) was added. The tube was mixed gently and returned to the incubator. 1 o'clock After a period of time, the labeled cells were washed five times by suspension in PBS and then After that, the cells were pelleted by centrifugation at 120×g for 5 minutes. Specific activity is per 10b cells. It was 2.7μC4.

E. Cytotoxicity test The design of the extended three-step cytotoxicity assay was performed as previously described (Ziegler et al. Ztegler-Hei tbrock et al. (1984) Journal Sub-National Cancer Institute (J, Natl. Cancer Inc.) Volume 72, page 23 and Colotta (1984), Journal of Sub-Immunology (J, Immunol, ) Volume 132, page 936), and it is shown diagrammatically in panel A of Figure 1. Indicated.

stage! (Briming); PBM in culture medium from 2X10'/wIl to 6. Serially diluted to 25xlO' cells/me and diluted with 50 microliters. microplates (μm) were placed in round-bottomed wells of a 966 microplate. blank ・Well size is 50 μ without PBM! Add the culture medium, and then Add 50 IJ1 of a 1% sodium lauryl sulfate aqueous solution to the "cis" well. I got it.

At this stage, TFNG is added to a certain concentration of IFN-G to reach the final concentration specified. Incorporated maleta (e.g. Eva, 525 U/m j! (7) Add 20 μff of medium containing I FN-G to 50 μl of PBM, 70 μl of PBM containing 150 U/μl of FN-G was obtained), and separately, IFN-G If the dose is kept constant, load 50 μl of cells in culture medium into the microwells. 150 U/μg of IFN-G was added directly to the PBM before treatment. plate centrifuged at 40 g for 3 min, then incubated under air containing 5% CO for 48 h. Incubated at 37°C.

Unless otherwise stated, all studies used recombinant IPNG (rI) for priming. F　N　G　4A; Amgen (A+mger+)　) was used. In other studies, Natural I FN G (nl FN G) has substantially the same modal stage n (1 - rigging); at specified time points, add 50 to IOQμ into wells containing PBM. Add the lymphin-containing medium or the cytokine preparation, and The plates were returned to the incubator and incubated for 6 hours. triggering reagent The final concentration of was calculated using the volume present during this stage of the assay.

Stage ■ (degradation): This degradation step involves adding 50 μl of 5l (r-labeled target) to each well. Started by adding cells (2X 10'/-1). of this plate After centrifugation at 40 g for 3 minutes and incubation and maintenance for 6 hours, the Remove one-half of the supernatant medium and place it in a scintillation counter. On Umu Channel, 2...! Counts were performed for 1 minute in Aquasol. Serum-free medium In a typical ground study, the background emissions are 2X10'/min. count, and the total count output was about 20 x 10'' CPM.

Result is, It is expressed as a percentage specific release capacity defined as .

Here, the CPM study is the count value from the well in question, and the CPM bank ground is Average counts from wells without PBM and CPM)-tals containing SDS. This is the average count from wells containing target cells. In general, CPM One blank “well used to secure the connection ground was All components used in the study wells (IFN-G and/or lymph) except the Normally, supernatant medium (LK-3N) containing lymphin is also used. , 70 kd cytotoxin, and in these environments, CP The M background includes counts directly released by this cytotoxin. (which does not account for more than 15% of the total count), IL-1 Alpha and Purified cytokines, including beta and beta, have direct cytotoxic effects on this target. It had no sexuality. To calibrate both halves of the chromatography, a “blank” Set up wells for each fraction to generate a small CPM bank ground. This was used to control fluctuations. In addition, this C5 assay method was performed without FBS and the same results were obtained. I got similar results.

All conditions are set three or four times, and two of them are usually mutually exclusive. The difference was within 10%. Results are expressed as mean: standard deviation.

F, Lymphin, cytokines and antibodies natural human I FN-G (nI FN-G) (0.83X 10”U/1m1) is Interferon Scie Interferon 5 Sciences (Ninny Blanc, NJ) 0 recombinant human obtained from Zoo I-/Ri (New Brunswick) IFN　G (rlFN　G”) (1,6X10’U/■) is Amgen (Aa+ gen) (Thousand Oaks, CA) Obtained from. Natural interleukin-2 (IL-2) (n1L-2) is Electronucleonics 5 Inc. ) Silver Springs, CVD It was found to contain endotoxin.

Recombinant IL-2 (rlL-2) (98 percent purity by HPLC) and recombinant C3F-1 (rC3F-1) is a product of Cetus Cozp. Dr. Peter Ra1ph (Emeryville, CA) ryville)). C3F-1 radioimmunoassay The RIA was kindly performed by Dr. Ralph. natural The interleukin (IL-1) (beta) is manufactured by Genzyme. Corp. (Boston, MA) or Systron Tech. C4sC45tronTechnolo (Pine Blue, NJ) (Pinebrook). Recombinant TL-1 (rIL -1) Alpha and beta were obtained from Genzyme (Genzyn+e).

Recombinant granule cell-monocyte colony activating factor (rGM-C3F) Genetic Institute 7-Dr. Steven C1ark (Cambridge, MA) 0 recombinant IIX necrosis factor alpha ( TNF-A) (Lonto 3056-63) (5,0xlO' U/■) and mouse ・Monoclonal anti-TNF-A (TNFD) (2,700 units per μg) Neutralizes TNF-A) is a product of Genetech, Inc. (South San Francisco, CA) ncisco) * The MIF test method was kindly provided by Briham & Mins Hospital (Brigham and Women'51 (ospit) Al) by Dr. Heinz Re5old (Hoston, MA) supernatant containing e-lymphoid The medium (LK-3N) is human PBM (3X10'/a+1! medium) and irradiation down. Co-culture B cells at a ratio of 3=1 or 5:1 (PBM:down) for 18-24 hours. It was prepared by To prepare control supernatant medium (Co-SN) , PBM and irradiated down cells were cultured separately and their supernatant media were mixed at a solid state. Ta.

LK-3N is 15-100 U/l1llI FN- as measured by RIA. G (CENTOCO, Malvern, PA) R)), 2-5U/gu! Lymphotoxin (Irvin, CA) e) University of California, G.A. Granger and R.S. , Yamamoto (Ya+*a+*oto) conducted assays on L929 cells. Measurement using thymidine incorporation by IL-2 dependent mouse T cell lineage 5~28U/+mi! of IL-2; of tissue factor in human monocytes Monocytoprocoagelant inducer assayed for its ability to induce expression ( MP IF) (Gregory et al. (1984) (Fed, Proc, Vol. 43, p. 1748) and sometimes target cells. On the other hand, it contained a 70kd cytotoxin with direct cytotoxicity. Co-3 N does not contain any of these activities, except for LK-3N excluding IFN-G. For this purpose, transfer 4 ml of supernatant to Affigel 10 at 4°C and at a flow rate of 3 ml/hour. Anti-human I FN-G mouse monochrome conjugated to (Affi-Get 10) 0.5 x 8, 5 am column of null antibody (La. Jolla), Scripps Clinic and Research Foundation (Scripps C11nic and Re5earch Foun claration); Robert Schreiber (courtesy of Dr. iber).

phorbol myristate acetate) (12-0-tetradecanoyl phorbol) 13-acetate; PMA: Sigma) and calcium Munophore A23187 (calimycin; CaI; Sigma-containing Nylon wool with supernatant medium (PMA/CaI SN) separated from PBM Prepared using non-adherent cells. 10 nanograms per milliliter of these cells of PM'A and 1 micromolar (μM) of A23187 calcium iono. The cells were suspended for 3 hours at 37°C in a medium containing fluorine. Then the cells The cells were washed three times and cultured for 48 hours in culture medium to produce lymphin-containing supernatant. Ta. The preparation PMA/Ca1-3N obtained from this operation has no detectable IFN G, IL2, does not contain lymphotoxin or direct cytotoxin However, it included MPIF. In addition, all PMA/C reported here Research on al-3N was conducted using a Sephadex G 100sf column chromatograph. performed using substantially pure CTF from the 55kd peak of the Raffy fraction. .

G, cloned human T lymphocytes PBM from normal (healthy, asymptomatic) volunteers was Allogeneic down lymphoblastoid cells irradiated (10,000 radians) and then cloned in soft agar in the presence of IL-2.

Gregory et al. (1985) Journal Subclinica Le Investigaci! (J, Cl1n, Invest,) Volume 76, 244 p. 0, the grown colonies were separated and as described by Gregory et al. (1985) Journal of Subclinical Investigation (J, Cl1n.

Invest, Vol. 76, p. 2440. in IL-2-containing liquid culture proboscis, with periodic reactivation by U-cells. Spread it out.

To generate supernatants containing Lymphin from TI3 cell clones, activation was performed for 3 weeks. cells that have not been exposed to IL-2 or have not received IL-2 for 5 days are washed, and I counted. Then, the clones selected in this study were irradiated to down cells and optimally The ratio of irradiated down cells to T cells is 4:1, which has been determined to be It was activated under the sea urchin condition. The resulting culture medium was sampled at various time points. Ko Control supernatant was obtained by culturing T cell clones and irradiated down cells separately, and then and the supernatant medium was mixed.

H0 molecular exclusion chromatography Amicon YM-10 membrane (10,000m, w, shielded Concentrate the medium supernatant containing Lymphin in 10 times by ultrafiltration using Dialyzed against endotoxin-free PBS (0.01M phosphate, pH 7.2), and Sephadex G 100 equilibrated with endotoxin-free PBS at 4°C. It was applied to a 2.5 x 90 cm column of sf. using PBS at a flow rate of 6 s1/h. Both fractions (4-l each) were eluted. For each fraction, bovine serum f-rubumin was used as a standard. Protein was assayed by the Lowry method using . Furthermore, the sample was 0.5U/ IFN-G using RIA of mJ anger level, and Gregory (Gregor (1985) Journal of Subclinical Investigation I reported in tion (J, Cl1n, Invest,) volume 76, page 2440. L-2-dependent thymidine incorporation into mouse T cell lines CTLL-4 and HT-2 IL-2 was assayed by quantification. Direct cytotoxin activity is Cytotoxicity was assayed in the absence of fector and target cells were tested in a standard 6-hour assay. It was determined by measuring 51(r) released from the cells.

! ,others All tests were performed three or four times, and the results were expressed as the mean plus or minus (± ) standard deviation was calculated. All media and reagents except those explicitly stated LiIIulus amoeboc Lysate assay method (St. Louis, MO) Jig? -Chemical Company (SigIIIa Chem, Co.), E-) Cusate ( E-toxate)) within detectable levels of 0.01 ng/ml. Contains no toxins. Phenotyping was performed at Raritan, NJ; Ortho Diagnosti Systems, Inc. ORTHO-MtiN manufactured by 5ysteas, Inc. E) Performed by indirect immunofluorescence method using a kit. These measurements Followed manufacturer's instructions.

The invention has been described in terms of preferred embodiments. Correction of published matters and or changes may be made without departing from the scope of the invention as described above. This will be obvious to those skilled in the art.

EAT E two-cho Day GURE 2 Toxicity (% specific release capacity) FIGURE 4 formula %formula% Cultivation time before harvesting the supernatant FIGURE 7 tube number 1. Indication of case PCT/US 8710107820 Title of invention Substantially pure Smart cytotoxic trigger factor 3, corrector Relationship to the case: Applicant 5. Date of amendment order: September 27, 1988 International Search Report

Claims (16)

[Claims] 1. Fractionation of proteins with apparent relative molecular weights of approximately 6 to 150 kilodaltons When measured by gel exclusion chromatography using a transparent gel matrix substantially pure human protein having an apparent molecular weight Mr of approximately 55 kilodaltons. A proteinaceous lymphokine, interferon gamma primed motor of tumor cells and killing factors including tumor necrosis factor alpha. It is possible to induce the secretion of interferon gamma and interferon. Ikin-2 and direct cytotoxins as well as the above pyrogen-free lymphoids. Cain. 2. An effective amount of substantially pure CTF is added to Primed Effect in an aqueous medium. of tumor cell killing factors including tumor necrosis factor alpha. Tumor cell enzymes, including tumor necrosis factor alpha, including initiating secretion A method of inducing peripheral blood mononuclear cells to secrete RING factor. 3. Claim No. 3 further includes a step of recovering the tumor necrosis factor alpha. The method described in Section 2. 4. Claims that the primed effector cell is a primed monocyte The method described in Scope No. 2. 5. 3. A method according to claim 2, wherein said addition is carried out in vitro. 6. Developing tumor necrosis factor alpha from human primed effector cells A virtually pure human drug that is effective in initiating the secretion of tumor cell-killing factors, including proteinaceous lymphokines in human primed ephemerase in aqueous medium. vector cells and an apparent relative of about 6 to 150 kilodaltons. Gel drainage using a gel matrix that can fractionate molecular weight proteins. The apparent molecular weight is approximately 55 kilodaltons as determined by dechromatography. and interferon gamma, interleukin-2 and direct cytokines. Like totoxin, it is a secreted tumor-killing factor that is virtually pyrogen-free. contacting the substantially pure lymphokine with the tumor cells to be killed. human primed effector cells that mediate tumor cell killing, including How to induce. 7. Claims in which the lymphokine is substantially non-glycosylated The method described in section 6. 8. Claim 6, wherein the effector cell is a primed monocyte. Method described. 9. The primed effector cell is a primed macrophage, Triggered in vitro and tumor necrosis factor alpha in the patient's tumor. The tumor cells that have been contacted with a tumor killing factor containing a 7. The method according to claim 6, wherein the cells are cells of a tumor in which mudomonocytes are present. 10. The effective triggering amount of the above lymphokines per milliliter of patient plasma , about 1 to about 100 units. 11. The effective triggering amount of the above lymphokines per kilogram of patient body weight is approximately 10. The method of claim 9, wherein the amount is from 60 to about 60,000 units. 12. Before the above addition step, the following steps: (a) the primed protein present in the tumor; Providing primed monocytes to the patient in addition to effector cells (b ) Maintain the patient long enough for added primed monocytes to concentrate in the tumor do, 10. The method of claim 9, further comprising: 13. The above-mentioned primed monocytes are subjected to the following steps: (i) The above-mentioned patients or obtaining a monocyte-containing leukocyte preparation from a donor; (ii) In vitro, the above preparation was treated with a priming amount of interferon gal. Mix with umma. (iii) The mixture thus prepared is primed with the monosites mentioned above. maintain for sufficient time, 13. The method according to claim 12, obtained by. 14. The primed effector cells are initiated in vitro and the tumor The tumor cells that have been brought into contact with the tumor cell killing factor are exposed to the outside within about 10 hours after the above initiation. 7. The method of claim 6, provided by: 15. The effective starting amount of the lymphokine is approximately About 3 to about 300 units per milliliter for 5 x 105 peripheral blood mononuclear cells. peripheral blood mononuclear cells and target tumor cells at a ratio of 50:1 and 10 Using 4 Act.D-treated WEHI164 cells, the above-mentioned peripheral 15. The method of claim 14, wherein blood mononuclear cells are primed. 16. Fractionation of proteins with apparent relative molecular weights from approximately 6 to approximately 150 kilodaltons determined by gel exclusion chromatography using a gel matrix that allows Substantially pure human protein with an apparent molecular weight Mr of approximately 14 kilodaltons Is it a qualitative lymphokine, interferon gamma primed monocyte? It induces the secretion of tumor cell-killing factors including tumor necrosis factor alpha. interferon gamma, interleukin-2 and direct support. Like Itotoxiso, the above-mentioned lymphokine is substantially free of pyrogens.